Functionalized zwitterionic and mixed charge polymers, related hydrogels, and methods for their use

ABSTRACT

Functionalized zwitterionic and mixed charge polymers and copolymers, methods for making the polymers and copolymers, hydrogels prepared from the functionalized zwitterionic and mixed charge polymers and copolymers, methods for making and using the hydrogels, and zwitterionic and mixed charge polymers and copolymers for administration for therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Patent Application No.62/048,155, filed Sep. 9, 2014, expressly incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Contract Nos. DMR1307375 and CBET-1264477, awarded by the National Science Foundation,and under Contract Nos. N00014-14-1-0090 and N00014-15-1-2277 awarded bythe Office of Naval Research. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Hydrogels have long been of interest for biological and biomaterialapplications due to their high water content that mimics theinterstitial tissue environment, ensures high diffusive permeability,and provides biomimetic mechanical strengths. Particular interest hasbeen given to PEG hydrogels and poly(2-hydroxyethyl methacrylate)(pHEMA) hydrogels because, in addition to the general properties ofhydrogels, they are also commonly considered to be low fouling,bioinert, and versatile.

pHEMA hydrogels have found use in and been studied for applications suchas contact lenses, artificial cornea, drug delivery vehicles, cartilagesubstitutes, and tissue scaffolds, among others. The hydration of pHEMA,however, is lower than that of native tissue, and its fouling, whilelow, is higher than other nonfouling materials. Furthermore, pHEMAfunctionalization via the hydroxyl group is generally difficult.

PEG hydrogels are routinely used, and can only be modified forapplications that require a bioinert background with specific addedbioactive functionalities for controlled in vitro and in vivo uses whenadditional functional groups are introduced into PEG hydrogels. However,it has been found that PEG is susceptible to oxidation. Thesusceptibility of PEG to oxidative damage reduces its utility forapplications that require long-term material stability. For applicationsin which maximal biological stability and nonfouling are required,however, PEG-based materials are insufficient.

Recently, zwitterionic compounds, including poly(carboxybetainemethacrylate), have been demonstrated to be ultra-low-fouling, meaningthat surfaces coated with these polymers allow less than 5 ng/cm²protein adsorption. Because of the high hydration and ultralow foulingproperties of zwitterionic materials, zwitterionic hydrogels are ofinterest as hydrogels with superior suitability for biomedicalapplications. The zwitterionic hydrogels studied so far, however, haveshown low mechanical strength, which limits their potential biologicaluses.

A need therefore exists for hydrogels having improved mechanicalproperties. The present invention seeks to fulfill this need andprovides further related advantages.

SUMMARY OF THE INVENTION

The present invention provides functionalized zwitterionic and mixedcharge polymers and copolymers, methods for making the polymers andcopolymers, hydrogels prepared from the functionalized zwitterionic andmixed charge polymers and copolymers, methods for making the hydrogels,methods for using the hydrogels for in vitro and in vivo cell culture,and zwitterionic and mixed charge polymers and copolymers foradministration for therapeutic agents.

Functionalized Zwitterionic Polymers and Mixed Charge Copolymers

In one aspect, the invention provides functionalized zwitterionicpolymers and copolymers and functionalized mixed charge copolymers.

In one embodiment, the invention provides a functionalized polymer,comprising a core having two or more polymeric branches covalentlycoupled to and extending from the core, wherein the polymeric branchescomprises constitutional units selected from the group consisting ofzwitterionic constitutional units and mixed charge constitutional units,and wherein the two or more polymeric branches each comprise one or morefunctional groups effective for covalently coupling the polymer to amaterial capable of forming a covalent bond with the one or morefunctionalize groups.

In certain embodiments, the polymer comprises three, four, five, or sixpolymeric branches. The number of branches can be varied and depends inthe nature of the core and the branches themselves. In some embodiments,the branches are branched.

In certain embodiments, the constitutional units are zwitterionicconstitutional units. In other embodiments, the constitutional units aremixed charge constitutional units. It will be appreciated that thezwitterionic polymers and mixed charge copolymers may further includeother constitutional units (e.g., the zwitterionic polymers can becopolymers). Suitable other constitutional units include constitutionalunits that include functional groups for imparting reactivity to thecopolymers, or other groups to impart desired properties to thecopolymer (e.g., anionic groups, cationic groups, neutral groups,hydrophobic groups, hydrophilic groups).

In certain embodiments, the functional group is positioned at theterminus of the polymeric branch. In other embodiments, the functionalgroup is positioned along the backbone of the polymeric branch. In someembodiments, one or more of the constitutional units comprise thefunctional group. The number of functional groups in the polymer or thepolymer branch can be controlled to achieve the desired overallfunctionality of the polymer or branch and will depend on the polymersultimate use.

In certain embodiments, the functional group is a thiol. In otherembodiments, the functional group is one of a reactive pair. In theseembodiments, the functional group is one of a reactive pair selectedfrom an azide and an alkyne, an azide and an alkene, a thiol and amaleimide, or a thiol and a dissulfide.

Zwitterionic and Mixed Charge Polymeric Hydrogels

In another aspect of the invention, zwitterionic and mixed chargehydrogels are provided.

In one embodiment, the hydrogel comprises a first polymer covalentlycoupled to a second polymer, wherein the first polymer comprises a firstcore having two or more polymeric branches covalently coupled to andextending from the first core, wherein the polymeric branches comprisefirst constitutional units selected from the group consisting ofzwitterionic constitutional units and mixed charge constitutional units,and wherein the two or more polymeric branches each comprise one or morefirst functional groups effective for covalently coupling the firstpolymer to the second polymer, wherein the second polymer comprises asecond core having two or more polymeric branches covalently coupled toand extending from the second core, wherein the polymeric branchescomprise second constitutional units selected from the group consistingof zwitterionic constitutional units and mixed charge constitutionalunits, and wherein the two or more polymeric branches each comprise oneor more second functional groups effective for covalently coupling thesecond polymer to the first polymer; and wherein the hydrogel comprisescovalent bonds linking the first and second polymers formed by reactionof the first and second functional groups.

In another embodiment, the hydrogel comprises a first polymer covalentlycoupled to a second polymer, wherein the first polymer comprises a firstcore having two or more polymeric branches covalently coupled to andextending from the first core, wherein the polymeric branches comprisefirst constitutional units selected from the group consisting ofzwitterionic constitutional units and mixed charge constitutional units,and wherein the two or more polymeric branches each comprise one or morefirst functional groups effective for covalently coupling the firstpolymer to the second polymer, wherein the second polymer comprises asecond core having two or more polymeric branches covalently coupled toand extending from the second core, wherein the polymeric branchescomprise second constitutional units selected from the group consistingof zwitterionic constitutional units and mixed charge constitutionalunits, and wherein the two or more polymeric branches each comprise oneor more second functional groups effective for covalently coupling thesecond polymer to the first polymer; and wherein the hydrogel comprisescovalent bonds linking the first and second polymers via a crosslinkingagent having two or more third functional groups, wherein the covalentbonds linking the first and second polymers are formed by reaction ofthe first and third functional groups and the second and thirdfunctional groups.

In certain of the hydrogel embodiments that include crosslinks formed bya crosslinking agent, it will be appreciated that the nature ofcrosslinking can be varied. In certain embodiments, the first and secondpolymers are different, have different functional groups, and arecrosslinked by the crosslinking agent with suitably reactive functionalgroups. In other embodiments, the first and second polymers are thesame, have the same functional groups, and are crosslinked by thecrosslinking agent with suitably reactive functional groups.

In certain of the hydrogel embodiments that include crosslinks formed bya crosslinking agent, the first and second functional groups are thesame. In certain of the hydrogel embodiments that include crosslinksformed by a crosslinking agent, the first functional group is a thiol,the second functional group is a thiol, and the third functional groupis a thiol or a dissulfide. In other of the hydrogel embodiments thatinclude crosslinks formed by a crosslinking agent, the first and thirdfunctional groups and the second and third functional groups are clickchemistry reactive pairs. In certain of these embodiments, the first andthird functional groups are selected from an azide and an alkyne, anazide and an alkene, a thiol and a maleimide, or a thiol and adissulfide. In certain of these embodiments, the second and thirdfunctional groups are selected from an azide and an alkyne, an azide andan alkene, a thiol and a maleimide, or a thiol and a dissulfide.

For the hydrogels of the invention as described above, in certainembodiments, the first polymer comprises three, four, five, or sixpolymeric branches. In certain embodiments, the first constitutionalunits are zwitterionic constitutional units. In other embodiments, thefirst constitutional units are mixed charge constitutional units.

In certain embodiments, the first functional groups are positioned atthe terminus of the polymeric branch. In other embodiments, the firstfunctional groups are positioned along the backbone of the polymericbranch. In certain embodiments, the first constitutional units comprisethe first functional group.

In certain embodiments, the second polymer comprises three, four, five,or six polymeric branches. In certain embodiments, the secondconstitutional units are zwitterionic constitutional units. In otherembodiments, the second constitutional units are mixed chargeconstitutional units.

In certain embodiments, the second functional groups are positioned atthe terminus of the polymeric branch. In other embodiments, the secondfunctional groups are positioned along the backbone of the polymericbranch. In certain embodiments, the second constitutional units comprisethe second functional group.

In certain embodiments, the first and second functional groups are thesame. In certain embodiments, the first functional group is a thiol andsecond functional group is a thiol. In other embodiments, the first andsecond functional groups are different. In certain embodiments, thefirst and second functional groups are a click chemistry reactive pair.Representative pairs include an azide and an alkyne, an azide and analkene, a thiol and a maleimide, or a thiol and a dissulfide.

The hydrogel of the invention may advantageously include additionalcomponents. In certain embodiment, the hydrogels further include cells,viruses, bacteria, and components thereof, or their genetically alteredvariants.

Representative cells include natural cells and their geneticallymodified counterparts, such as exocrine secretory epithelial cells,hormone secreting cells, keratinizing epithelial cells, wet stratifiedbarrier epithelial cells, sensory transducer cells, autonomic neuroncells, sense organ and peripheral neuron supporting cells, neurons andglial cells, lens cells, hepatocytes, adipocytes, lipocytes, barrierfunction cells, kidney cells, heart cells, extracellular matrix cells,contractile cells, blood and immune system cells such as erythrocytes,monocytes, neutrophils, mast cells, T cells, stem cells, germ cells,nurse cells, interstitial cells, progenitor cells, hematopoietic stemcells.

Representative viruses include DNA viruses, RNA viruses, reversetranscriptase viruses, retroviruses, and adenoviruses.

Representative bacteria include pathogenic bacteria, Gram-positivebacteria, Gram-negative bacteria, cyanobacteria. Representative bacteriaspecies include clostridial species, S. typhimurium, and E. coli.

In certain embodiments, the hydrogels of the invention includeoligonucleotides (DNAs and RNAs), lipoplexes, polymersomes, polyplexes,dendrimers, inorganic particles.

In other embodiments, the hydrogels of the invention include proteins,peptides, polysaccharides, or small molecules.

In certain embodiments, the hydrogels of the invention include atherapeutic or diagnostic agent.

In other embodiments, the hydrogels of the invention include ananomaterial.

Surfaces Treated and Devices Made with Zwitterionic and Mixed ChargePolymeric Hydrogels

In a further aspect, the invention provides substrates treated ahydrogel of the invention. In one embodiment, the invention provides asubstrate having a surface, wherein at least a portion of the surface iscoated with the hydrogel. In certain embodiments, the surface isentirely coated with the hydrogel.

In a related aspect, the invention provides devices (e.g., medicaldevices) formed at least in part from the hydrogels of the invention ordevices (e.g., medical devices) that incorporate the hydrogels of theinvention. In certain of these embodiments, the devices are madeentirely or partially from the hydrogels of the invention.

Representative substrates and devices include the following: implantablebiosensor, wound care device, sealant, contact lens, dental implant,orthopedic device (artificial joint, artificial bone, artificialligament, artificial tendon), cardiovascular device (cathether,artificial valve, artificial vessel, artificial stent, LVAD, rhythmmanagement device), gastroenterology device (feeding tube, alimentarycanal clip, gastro-intestinal sleeve, gastric balloon), OB/Gyn device(implantable birth control device, vaginal sling), nephrology device(anastomotic connector, subdermal port), neurosurgery device (nerveguidance tube, cerebrospinal fluid drain or shunt), dermatology device(skin repair device), ophthalmic device (shunt), otorhinolaryngologydevice (stent, cochlear implant, tube, shunt, spreader), intra-ocularlens. aesthetic implant (breast implant, nasal implant, cheek implant),neurologic implant (nerve stimulation device), cochlear implant, nerveconduit, hormone control implant (blood sugar sensor, insulin pump),implanted biosensor, access port device, tissue scaffold pulmonic device(valve for management of COPD or artificial lungs), radiology device(radio-opaque or sono-opaque markers), or urology device (catheter,artificial urethrae).

Zwitterionic and Mixed Charge Polymeric Hydrogels Uses

In another aspect, the invention provides uses for the zwitterionic andmixed charge polymeric hydrogels.

In certain embodiments, the invention provides a medium for protection,preservation, or growth of cells comprising the hydrogel. The medium canfurther include one or more nutrients or growth factors. The medium canbe used in vitro or in vivo.

In certain embodiments, the invention provides a surgical sealantcomprising the hydrogel.

In certain embodiments, the invention provides a surgical anti-adherencecoating comprising the hydrogel.

In certain embodiments, the invention provides a surgical fillercomprising the hydrogel.

In certain embodiments, the invention provides a wound dressingcomprising the hydrogel.

In certain embodiments, the invention provides an aesthetic fillercomprising the hydrogel.

In certain embodiments, the invention provides an aesthetic fillerpre-formed to a specific shape comprising the hydrogel.

In certain embodiments, the invention provides an orthopedic soft tissuereplacement (e.g., for cartilage or spinal discs) comprising thehydrogel.

In certain embodiments, the invention provides a tissue growth scaffold,comprising the hydrogel. The scaffold can be used in vitro or in vivo.

In certain embodiments, the invention provides a medical device formedat least in part from a hydrogel.

In certain embodiments, the invention provides a medical deviceincorporating a hydrogel.

Methods for Making Zwitterionic and Mixed Charge Polymeric Hydrogels

In a further aspect, the invention provides methods for makingzwitterionic and mixed charge polymeric hydrogels.

In a first embodiment, the invention provides a method for forming ahydrogel, comprising reacting a first polymer with a second polymer toprovide a hydrogel, wherein the first polymer comprises a first corehaving two or more polymeric branches covalently coupled to andextending from the first core, wherein the polymeric branches comprisefirst constitutional units selected from the group consisting ofzwitterionic constitutional units and mixed charge constitutional units,and wherein the two or more polymeric branches each comprise one or morefirst functional groups effective for covalently coupling the firstpolymer to the second polymer, wherein the second polymer comprises asecond core having two or more polymeric branches covalently coupled toand extending from the second core, wherein the polymeric branchescomprise second constitutional units selected from the group consistingof zwitterionic constitutional units and mixed charge constitutionalunits, and wherein the two or more polymeric branches each comprise oneor more second functional groups effective for covalently coupling thesecond polymer to the first polymer; and wherein the hydrogel is formedby covalent bond formation between the first and second functionalgroups.

In certain embodiments, the hydrogel is formed in situ (e.g., in vivo).In other embodiments, the hydrogel is formed in a vessel. In eitherembodiment, the hydrogel can be used for cell culture.

In a second embodiment, the invention provides a method for forming ahydrogel in vivo, comprising:

(a) disposing a first polymer at a site in vivo; and

(b) disposing a second polymer at the site, whereby the second polymercontacts the first polymer at the site to provide a hydrogel,

wherein the first polymer comprises a first core having two or morepolymeric branches covalently coupled to and extending from the firstcore, wherein the polymeric branches comprise first constitutional unitsselected from the group consisting of zwitterionic constitutional unitsand mixed charge constitutional units, and wherein the two or morepolymeric branches each comprise one or more first functional groupseffective for covalently coupling the first polymer to the secondpolymer,

wherein the second polymer comprises a second core having two or morepolymeric branches covalently coupled to and extending from the secondcore, wherein the polymeric branches comprise second constitutionalunits selected from the group consisting of zwitterionic constitutionalunits and mixed charge constitutional units, and wherein the two or morepolymeric branches each comprise one or more second functional groupseffective for covalently coupling the second polymer to the firstpolymer; and

wherein the hydrogel is formed by covalent bond formation between thefirst and second functional groups.

In certain embodiments, the first polymer and second polymer aredisposed at the site by injection, spray, pouring, and dipping.

In a third embodiment, the invention provides a method for forming ahydrogel, comprising reacting a first polymer, a second polymer, and acrosslinking agent to provide a hydrogel, wherein the first polymercomprises a first core having two or more polymeric branches covalentlycoupled to and extending from the first core, wherein the polymericbranches comprise first constitutional units selected from the groupconsisting of zwitterionic constitutional units and mixed chargeconstitutional units, and wherein the two or more polymeric brancheseach comprise one or more first functional groups effective forcovalently coupling the first polymer to the crosslinking agent, whereinthe second polymer comprises a second core having two or more polymericbranches covalently coupled to and extending from the second core,wherein the polymeric branches comprise second constitutional unitsselected from the group consisting of zwitterionic constitutional unitsand mixed charge constitutional units, and wherein the two or morepolymeric branches each comprise one or more second functional groupseffective for covalently coupling the second polymer to the crosslinkingagent; wherein the crosslinking agent comprises two or more thirdfunctional groups effective for covalently coupling the first polymer tothe second polymer by forming crosslinks between the first and secondpolymers; and wherein the hydrogel is formed by covalent bond formationbetween the first and third functional groups and the second and thirdfunctional groups.

In certain embodiments of the third embodiment, the first and secondfunctional groups are the same; the first functional group is a thiol,the second functional group is a thiol, and the third functional groupis a thiol or a disulfide; and the first and third functional groups andthe second and third functional groups are click chemistry reactivepairs. In certain embodiments, the first and third functional groups areselected from an azide and an alkyne, an azide and an alkene, a thioland a maleimide, or a thiol and a dissulfide. In certain embodiments,the second and third functional groups are selected from an azide and analkyne, an azide and an alkene, a thiol and a maleimide, or a thiol anda dissulfide.

In the above methods (first, second, and/or third embodiments), thefirst polymer comprises three, four, five, or six polymeric branches.

In certain of these embodiments, the first constitutional units arezwitterionic constitutional units. In others of these embodiments, thefirst constitutional units are mixed charge constitutional units.

In certain embodiments, the first functional groups are positioned atthe terminus of the polymeric branch. In other embodiments, the firstfunctional groups are positioned along the backbone of the polymericbranch. In certain embodiments, one or more of the first constitutionalunits comprise the first functional group.

In the above methods (first, second, and/or third embodiments), thesecond polymer comprises three, four, five, or six polymeric branches.

In certain of these embodiments, the second constitutional units arezwitterionic constitutional units. In others of these embodiments, thesecond constitutional units are mixed charge constitutional units.

In certain embodiments, the second functional groups are positioned atthe terminus of the polymeric branch. In other embodiments, the secondfunctional groups are positioned along the backbone of the polymericbranch. In certain embodiments, one or more of the second constitutionalunits comprise the second functional group.

In certain embodiments, the first and second functional groups are thesame. In certain embodiments, the first functional group is a thiol andsecond functional group is a thiol.

In other embodiments, the first and second functional groups aredifferent. In certain embodiments, the first and second functionalgroups are a click chemistry reactive pair. In certain embodiments, thefirst and second functional groups are selected from an azide and analkyne, an azide and an alkene, a thiol and a maleimide, or a thiol anda dissulfide.

Zwitterionic and Mixed Charge Star Polymer Therapeutic Agent Conjugates

In another aspect, the invention provides zwitterionic and mixed chargestar polymer therapeutic agent conjugates.

In one embodiment, the invention provides a polymer, comprising a corehaving two or more polymeric branches covalently coupled to andextending from the core, wherein the polymeric branches comprisesconstitutional units selected from the group consisting of zwitterionicconstitutional units and mixed charge constitutional units, and whereinthe zwitterionic constitutional units and the mixed chargeconstitutional units comprise a therapeutic agent covalently coupledthereto.

In certain embodiments, the therapeutic agent is covalently coupled tothe constitutional unit by a hydrolyzable bond.

In certain embodiments, the polymer comprises three, four, five, or sixpolymeric branches. In certain embodiments, the constitutional units arezwitterionic constitutional units. In other embodiments, theconstitutional units are mixed charge constitutional units.

In a related aspect, a method for administering a therapeutic agent to asubject is provided. In the method, a therapeutically effective amountof a polymer of the invention having a therapeutic agent covalentlycoupled thereto is administered to a subject (e.g., a warm-bloodedanimal, such as a human) in need thereof.

In certain embodiments, administering the polymer comprises systemic,topical, or local administration. In certain embodiments, administeringthe polymer comprises inhalation, oral, and transdermal administration.In certain embodiments, administering the polymer comprises intravenousinjection, intramuscular injection, subcutaneous injection,intraperitoneal injection, or local injection.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIG. 1 is a schematic illustration of the synthesis of a representativestar polymer of the invention having zwitterionic (polycarboxybetaine)branches. In this star polymer the branches are shown terminating with abromomethyl group, which is the radical initiator group for continuedpolymerization (e.g., atom transfer radical polymerization, ATRP). Thestar polymer was prepared by ATRP from the tetrafunctional core havingthe illustrated radical initiator groups (—O(C═O)—C(CH₃)₂—Br) andcarboxybetaine methacrylate (CH₂═C(CH₃)—C(═O)—CH₂CH₂—N(CH₃)₂⁺—CH₂CH₂—CO₂ ⁻, CBMA) monomers.

FIG. 2 is a schematic illustration of the synthesis of a representativestar polymer of the invention having zwitterionic (polycarboxybetaine)branches terminated with a thiol group (—CH₂—SH). This star polymer wasprepared from the star polymer illustrated in FIG. 1 by click chemistry(reaction of the star polymer with sodium azide and 3-thioacetylene).

FIGS. 3A and 3B schematically illustrate the preparation of arepresentative hydrogel of the invention prepared from suitablyfunctionalized star polymers of the invention. FIG. 3A illustrates thesyntheses of two representative star polymers of the invention: (a) astar polymer having zwitterionic (polycarboxybetaine) branchesterminated with a thiol group (—CH₂—SH) denoted pCB-A; and (b) a starpolymer having zwitterionic (polycarboxybetaine) branches terminatedwith a pyridyl disulfide group (—CH₂—S—S—C₅H₄N) denoted pCB-B. Thesestar polymers were prepared from the star polymer illustrated in FIG. 1by click chemistry (reaction of the star polymer with sodium azide and(a) 3-thioacetylene or (b) 3-pyridyl disulfide acetylene). FIG. 3Billustrates the preparation of representative hydrogels of the inventionprepared from reaction of the star polymers (pCB-A and pCB-B)illustrated in FIG. 3A. The hydrogels can be formed by simply mixingpCB-A with pCB-B in the presence of cells.

FIGS. 4A-4D present data for encapsulated islets cells cultured in arepresentative hydrogel (pCB hydrogel) of the invention. FIG. 4A is animage showing the results of a LIVE/DEAD assay performed on encapsulatedislets within pCB hydrogels after 18-day in vitro culture. Scale bar:300 μm. Effects of hydrogel encapsulation of islet cells is shown inFIGS. 4B-4D. FIG. 4B compares glucose consumption over time for isletcells cultured in a representative hydrogel of the invention (pCB) andcontrol (Ctrl) (n=3). FIG. 4C compares insulin secretion in staticincubation on day 18 (n=4) for islet cells cultured in a representativehydrogel of the invention (pCB) and control (Ctrl). FIG. 4D comparesdynamic insulin release on day 18 for islet cells cultured in arepresentative hydrogel of the invention (pCB) and control (Ctrl) asassessed by sequential stimulating by low (3 mM, blue arrow), high (17mM, red arrow), and low (3 mM) glucose media again using a perifusionsystem (n=3). In the figures, * indicates significant difference fromcontrol islets (without hydrogel, p<0.05). Mean±SEM.

FIGS. 5A and 5B present data for encapsulated peripheral blood stem cell(PBSC) cells cultured in a representative hydrogel (pCB hydrogel) of theinvention. FIG. 5A illustrates hydrogel modulus over time (hydrogeldegradation). FIG. 5B is an image showing the results of a LIVE/DEADassay performed on encapsulated PBSC cells within the pCB hydrogelsafter 7-day in vitro culture.

FIG. 6 is a schematic illustration of the release of a representativetherapeutic agent from a representative star polymer of the inventionhaving zwitterionic (polycarboxybetaine) branches. The star polymer wasprepared by ATRP from the tetrafunctional core having the radicalinitiator groups and carboxybetaine methacrylate-therapeutic agentmonomers (CH₂═C(CH₃)—C(═O)—CH₂CH₂—N(CH₃)₂ ⁺—CH₂CH₂—CO₂-therapeuticagent). In the representative star polymer shown, the branches arefurther extended by extension polymerization with CBMA to providebranches having constitutional units that are zwitterionic in additionto constitutional units bearing the therapeutic agent. The star polymerillustrated in FIG. 6 is a block copolymer. It will be appreciated thatin certain embodiments, the star polymer is a random copolymer preparedby copolymerization of zwitterionic monomers (e.g., CBMA) andzwitterionic-therapeutic agent monomers (e.g., CBMA-therapeutic agent).On hydrolysis of the star polymer, the therapeutic agent is released.Hydrolysis of the star polymer and release of the therapeutic agentregenerates a zwitterionic constitutional unit in the star polymer. Asshown in FIG. 6, the therapeutic agent is ibuprophen, and the monomerfor synthesizing the star polymer capable of releasing ibuprophen isprepared by condensation of CBMA with ibuprofen.

FIG. 7 is a schematic illustration of the preparation of arepresentative star pCB of the invention useful for making hydrogels forcell encapsulation.

FIG. 8 is compares viability of T cells encapsulated withinrepresentative pCB hydrogels of the invention.

FIG. 9 shows poly(carboxybetaine) polymers functionalized withcomplimentary clickable groups R and R* (steps 1 a and 1 b) and theresulting hydrogel formed by their mixing.

FIG. 10 depicts poly(carboxybetaine) copolymers functionalized withcomplimentary clickable groups R and R* (steps 2 a and 2 b) bycopolymerization and the resulting hydrogel formed by their mixing.

FIG. 11 depicts end-group functionalized polymers and hydrogel formedfrom the polymers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides zwitterionic and mixed charge starpolymers and copolymers, functionalized zwitterionic and mixed chargestar polymers and copolymers useful for therapeutic agent delivery andhydrogel formations, zwitterionic and mixed charge star polymers andcopolymers having therapeutic agents covalently coupled thereto fortherapeutic agent delivery, and hydrogels prepared from thefunctionalized zwitterionic and mixed charge star polymers.

As used herein, the term “zwitterionic polymer or copolymer” refers to apolymer or copolymer having zwitterionic constitutional units.Zwitterionic constitutional units have pendant groups (i.e., groupspendant from the polymer backbone) that include zwitterionic groups.Representative zwitterionic pendant groups include carboxybetaine groups(e.g., —R_(a)—N⁺(R_(b))(R_(c))—R_(d)—CO₂ ⁻, where R_(a) is a linkergroup that covalently couples the polymer backbone to the cationicnitrogen center of the carboxybetaine groups, R_(b) and R_(c) arenitrogen substituents, R_(d) is a linker group that covalently couplesthe cationic nitrogen center to the carboxy group of the carboxybetainegroup).

The term “mixed charge copolymer” refers to a copolymer havingsubstantially equal numbers of positively charged constitutional unitsand negatively charged constitutional units to provide a copolymer thatis substantially electronically neutral. In certain embodiments, themixed charge copolymer are random copolymers that do not have extensiveregions along the polymer backbone that are positively charged ornegatively charged (i.e., the positively and negatively chargedconstitutional units are relatively uniformly distributed along thepolymer backbone). Representative mixed charge pendant groups includecarboxy groups (e.g., —R_(a)—CO₂ ⁻, where R_(a) is a linker group thatcovalently couples the carboxy group to the polymer backbone) and aminogroups (e.g., —R_(a)—N⁺(R_(b))(R_(c))(R_(d)), where R_(a) is a linkergroup that covalently couples the cationic nitrogen center to thepolymer backbone, and R_(b), R, and R_(d) are nitrogen substituents).

The term “star polymer or copolymer” refers to a branched polymer orcopolymer in which two or more polymer or copolymer branches extend froma core. Representative star polymers and copolymers of the inventioninclude two, three, four, five, six, or more branches extending from thecore. The core is a group of atoms having two or more functional groupsfrom which the branches can be extended by polymerization.Representative cores have two, three, four, five, six, or morefunctional groups from which the branches can be extended. In certainembodiments, the branches are zwitterionic polymeric or copolymericbranches. In other embodiments, the branches are mixed chargecopolymeric branches

The term “functionalized polymer or copolymer” refers to a polymer ofcopolymer that includes a functional group that renders to polymer orcopolymer reactive to covalent coupling to another polymer of copolymerthat is also a functionalized polymer or copolymer. In the practice ofthe invention, functionalized star polymers and copolymers of theinvention react through their functional groups to form covalent bondsthat covalently couple the polymers and copolymers (e.g., crosslink thepolymer and copolymers). To covalently couple a first functionalizedpolymer or copolymer to a second functionalized polymer or copolymer,the functional groups of the first and second polymers or copolymers(i.e., the first and second functional groups, respectively) havecomplimentary reactivity. The first and second functional groups arereactive pairs. In certain embodiments, the first and second functionalgroups react on mixing at a temperature between room temperature andphysiological temperature to form a bond (e.g., without externalstimulus). Suitable such reactive pairs are known in the art.Representative useful reactive pairs include thiol/maleimide and clickchemistry reactive pairs (e.g., azides/alkynes and azides/alkenes).

Polymer Definitions

The term “constitutional unit” refers to an atom or group of atoms in apolymer that includes a part of the polymer chain together with itspendant atoms or groups of atoms, if any. The constitutional unit canrefer to a repeat unit. The constitutional unit can also refer to an endgroup on a polymer chain. For example, the constitutional unit ofpolyethylene glycol can be —CH₂CH₂O— corresponding to a repeat unit, or—CH₂CH₂OH corresponding to an end group.

The term “repeat unit” corresponds to the smallest constitutional unit,the repetition of which constitutes a regular macromolecule (or oligomermolecule or block).

The term “end group” refers to a constitutional unit with only oneattachment to a polymer chain, located at the end of a polymer. Forexample, the end group can be derived from a monomer unit at the end ofthe polymer, once the monomer unit has been polymerized. As anotherexample, the end group can be a part of a chain transfer agent orinitiating agent that was used to synthesize the polymer.

The term “monomer” is a polymerizable compound that, on polymerization,contributes one or more constitutional units in the structure of thepolymer.

The term “polymer” refers to the product that is the result ofpolymerization of a single monomer.

The term “copolymer” refers to a polymer that is the result ofpolymerization of two or more different monomers. The number and thenature of each constitutional unit can be separately controlled in acopolymer. The constitutional units can be disposed in a purely random,an alternating random, a regular alternating, a regular block, or arandom block configuration unless expressly stated to be otherwise. Apurely random configuration can, for example, be:x-x-y-z-x-y-y-z-y-z-z-z . . . or y-z-x-y-z-y-z-x-x . . . . Analternating random configuration can be: x-y-x-z-y-x-y-z-y-x-z . . . ,and a regular alternating configuration can be: x-y-z-x-y-z-x-y-z . . ..

Functionalized Zwitterionic Polymers and Mixed Charge Copolymers

In one aspect, the invention provides functionalized zwitterionic andmixed charge polymers and copolymers. In certain embodiments, thefunctionalized zwitterionic and mixed charge polymers and copolymers ofthe invention are functionalized zwitterionic star and mixed charge starpolymers and copolymers.

As noted above, for the star polymers and copolymers of the invention,the branches can be any zwitterionic or mixed charge polymers and theirprecursors that can be converted to zwitterionic or mixed chargepolymers via hydrolysis, ultraviolet irradiation, or heat. Thezwitterionic or mixed charge polymers can be obtained by anypolymerization method effective for polymerization of unsaturatedmonomers, including atom transfer radical polymerization (ATRP),reversible addition-fragmentation chain-transfer polymerization (RAFT),photo-polymerization, ring-opening polymerization (ROP), condensation,Michael addition, branch generation/propagation reaction, or otherreactions. In certain embodiments, the polymers having terminalfunctional groups are able to specifically bind to a binding partner andat the same time avoid nonspecific biofouling, which is imparted to thepolymers by their zwitterionic or mixed charge structures. By virtue oftheir functionalized terminal ends, the polymers of the invention can beconverted to further functionalized polymers useful for making hydrogelsof the invention, by complimentary coupling chemistries (e.g., clickchemistries, thiol exchange reactions, reductive reactions, and otherchemistries known in the art). These functional end groups can be pre-and post-modified after the branches are created.

Zwitterionic Monomers.

In certain embodiments, the functionalized polymers, and copolymers ofthe invention are polymers prepared from polymerization of suitablepolymerizable zwitterionic monomers. In certain of these embodiments,the polymer or copolymer has repeating units having formula (I):

wherein

R₄ is selected from hydrogen, fluorine, trifluoromethyl, C1-C6 alkyl,and C6-C12 aryl groups;

R₅ and R₆ are independently selected from alkyl and aryl, or takentogether with the nitrogen to which they are attached form a cationiccenter;

L₄ is a linker that covalently couples the cationic center [N⁺(R₅)(R₆)]to the polymer backbone [—(CH₂—CR₄)_(n)—];

L₅ is a linker that covalently couples the anionic center [A₂(═O)O⁻] tocationic center;

A₂ is C, SO, SO₂, P, or PO;

n is an integer from 5 to about 10,000; and

* represents the point at which the repeating unit is covalently linkedto an adjacent repeating unit or a functional group useful for forminghydrogels.

In the polymer, the pendant zwitterionic groups are internal salts andM⁺ and X⁻ are absent.

In one embodiment, R₄ is C1-C3 alkyl.

R₅ and R₆ are independently selected from alkyl and aryl, or takentogether with the nitrogen to which they are attached form a cationiccenter. In one embodiment, R₅ and R₆ are C1-C3 alkyl.

In certain embodiments, L₄ is selected from the group consisting of—C(═O)O—(CH₂)_(n)— and —C(═O)NH—(CH₂)_(n)—, wherein n is an integer from1 to 20. In certain embodiments, L₄ is —C(═O)O—(CH₂)_(n)—, wherein n is1-6.

In certain embodiments, L₅ is —(CH₂)_(n)—, where n is an integer from 1to 20.

In certain embodiments, A₂ is C or SO.

In certain embodiments, n is an integer from 5 to about 5,000.

In one embodiment, R₄, R₅, and R₆ are methyl, L₄ is —C(═O)O—(CH₂)₂—, L₅is —(CH₂)—, A₁ is C, and n is an integer from 10 to about 1,000.

The zwitterionic polymers and copolymers of the invention can beprepared by polymerization of monomers having formula (II):CH₂═C(R₄)-L₄-N⁺(R₅)(R₆)-L₅-A₂(═O)O⁻  (II)wherein R₄, R₅, R₆, L₄, L₅, and A₂, are as described above for therepeating unit of formula (I).

Representative zwitterionic polymer branches of the invention haveformula (III):PB-(L₄-N⁺(R₅)(R₆)-L₅-A₂(═O)O⁻)_(n)  (III)wherein R₅, R₆, L₄, L₅, A₂, and n are as described above for therepeating unit of formula (I), and PB is the polymer backbone thatincludes repeating units [formula (I)].

Mixed Charge Comonomers.

In another aspect, the invention provides mixed charge copolymersprepared from copolymerization of ion pair comonomers. As noted above,the mixed charge copolymers having a polymer backbone, a plurality ofpositively charged repeating units, and a plurality of negativelycharged repeating units. In the practice of the invention, thesecopolymers may be prepared by polymerization of ion-pair comonomers.

The mixed charge copolymer includes a plurality of positively chargedrepeating units, and a plurality of negatively charged repeating units.In one embodiment, the mixed charge copolymer is substantiallyelectronically neutral. As used herein, the term “substantiallyelectronically neutral” refers to a copolymer that imparts advantageousnonfouling properties to the copolymer. In one embodiment, asubstantially electronically neutral copolymer is a copolymer having anet charge of substantially zero (i.e., a copolymer about the samenumber of positively charged repeating units and negatively chargedrepeating units). In one embodiment, the ratio of the number ofpositively charged repeating units to the number of the negativelycharged repeating units is from about 1:1.1 to about 1:0.5. In oneembodiment, the ratio of the number of positively charged repeatingunits to the number of the negatively charged repeating units is fromabout 1:1.1 to about 1:0.7. In one embodiment, the ratio of the numberof positively charged repeating units to the number of the negativelycharged repeating units is from about 1:1.1 to about 1:0.9.

In one embodiment, the mixed charge copolymers are prepared fromcopolymerization of suitable polymerizable ion pair comonomers.

Representative ion-pair comonomers useful in the invention have formulas(IV) and (V):CH₂═C(R₇)-L₆-N⁺(R₉)(R₁₀)(R₁₁)X⁻  (IV)CH₂═C(R₈)-L₇-A₃(═O)—O⁻-M⁺  (V)

In this embodiment, the mixed charge copolymer has repeating unitshaving formula (VI):

wherein

R₇ and R₈ are independently selected from hydrogen, fluorine,trifluoromethyl, C1-C6 alkyl, and C6-C12 aryl groups;

R₉, R₁₀, and R₁₁ are independently selected from alkyl and aryl, ortaken together with the nitrogen to which they are attached form acationic center;

A₃(═O)OM) is an anionic center, wherein A₃ is C, SO, SO₂, P, or PO, andM is a metal or organic counterion;

L₆ is a linker that covalently couples the cationic center[N⁺(R₉)(R₁₀)(R₁₁)] to the polymer backbone;

L₇ is a linker that covalently couples the anionic center [A(═O)OM] tothe polymer backbone;

X⁻ is the counter ion associated with the cationic center;

n is an integer from 5 to about 10,000;

p is an integer from 5 to about 10,000; and

* represents the point at which the repeating units is covalently linkedto either and adjacent repeating unit or a functional group useful forforming hydrogels.

In one embodiment, R₇ and R₈ are C1-C3 alkyl.

R₉, R₁₀, and R₁₁ are independently selected from alkyl and aryl, ortaken together with the nitrogen to which they are attached form acationic center. In one embodiment, R₉, R₁₀, and R₁₁ are C1-C3 alkyl.

In certain embodiments, L₆ is selected from the group consisting of—C(═O)O—(CH₂)_(n)— and —C(═O)NH—(CH₂)_(n)—, wherein n is an integer from1 to 20. In certain embodiments, L₆ is —C(═O)O—(CH₂)_(n)—, wherein n is1-6.

In certain embodiments, L₇ is a C1-C20 alkylene chain. Representative L₇groups include —(CH₂)—, where n is 1-20 (e.g., 1, 3, or 5)

In certain embodiments, A₃ is C, S, SO, P, or PO.

In certain embodiments, n is an integer from 5 to about 5,000.

In one embodiment, R₇, R₈, R₉, R₁₀, and R₁₁ are methyl, L₆ and L₇ are—C(═O)O—(CH₂)₂—, A₁ is C, and n is an integer from 10 to about 1,000.

Representative mixed charge copolymer branches have formula (VII):PB-[L₆-N⁺(R₉)(R₁₀)(R₁₁)]_(n)[L₇-A₃(═O)—O⁻M⁺)]_(p)(X⁻)_(n)  (VII)wherein L₆, N⁺(R₉)(R₁₀)(R₁₁), L₇, A₃(═O)O-M⁺, X⁻, n, and p are asdescribed above, and PB is the polymer backbone that includes repeatingunits [formula (VI)].

The following is a description of the crosslinking agent, monomers,comonomers, polymers, copolymers, and crosslinks of formulas (I)-(VI)described above.

In the above formulas, PB is the polymer backbone. Representativepolymer backbones include vinyl backbones (e.g.,—C(R′)(R″)—C(R′″)(R″″)—, where R′, R″, R′″, and R′″ are independentlyselected from hydrogen, alkyl, and aryl) derived from vinyl monomers(e.g., acrylate, methacrylate, acrylamide, methacrylamide, styrene).Other suitable backbones include polymer backbones that provide forpendant groups. Other representative polymer backbones include peptide(polypeptide), urethane (polyurethane), and epoxy backbones.

Similarly, in the above formulas, CH₂═C(R)— is the polymerizable group.It will be appreciated that other polymerizable groups, including thosenoted above, can be used to provide the monomers and polymers of theinvention.

In the above formulas, N⁺ is the cationic center. In certainembodiments, the cationic center is a quaternary ammonium (e.g., Nbonded to L₄, R₅, R₆, and L₅). In addition to ammonium, other usefulcationic centers (R₅ and R₆ taken together with N) include imidazolium,triazaolium, pyridinium, morpholinium, oxazolidinium, pyrazinium,pyridazinium, pyrimidinium, piperazinium, and pyrrolidinium.

R₄, R₅, R₆, R₉, R₁₀, and R₁₁ are independently selected from hydrogen,alkyl, and aryl groups. Representative alkyl groups include C1-C10straight chain and branched alkyl groups. In certain embodiments, thealkyl group is further substituted with one of more substituentsincluding, for example, an aryl group (e.g., —CH₂C₆H₅, benzyl).Representative aryl groups include C6-C12 aryl groups including, forexample, phenyl. For certain embodiments of the above formulas, R₅ andR₆, or R₉, R₁₀, and R₁₁ are taken together with N⁺ form the cationiccenter.

L₄ (or L₆) is a linker that covalently couples the cationic center tothe polymer backbone. In certain embodiments, L₄ includes a functionalgroup (e.g., ester or amide) that couples the remainder of L₄ to thepolymer backbone (or polymerizable moiety for the monomers). In additionto the functional group, L₄ can include a C1-C20 alkylene chain.Representative L₄ groups include —C(═O)O—(CH₂)_(n)— and—C(═O)NH—(CH₂)_(n)—, where n is 1-20 (e.g., 3).

L₅ is a linker that covalently couples the cationic center to theanionic group (i.e., (A=O)O⁻). L₅ can be a C1-C20 alkylene chain.Representative L₅ groups include —(CH₂)_(n)—, where n is 1-20 (e.g., 1,3, or 5).

L₇ is a linker that covalently couples the polymer backbone to theanionic group. L₇ can be a C1-C20 alkylene chain. Representative L₇groups include —(CH₂)_(n)—, where n is 1-20 (e.g., 1, 3, or 5).

A(═O)—O⁻ is the anionic center. The anionic center can be a carboxylicacid ester (A is C), a sulfinic acid (A is SO), a sulfonic acid (A isSO₂), a phosphinic acid (A is P), or a phosphonic acid (A is PO).

In the above formulas, representative alkyl groups include C1-C30straight chain and branched alkyl groups. In certain embodiments, thealkyl group is further substituted with one of more substituentsincluding, for example, an aryl group (e.g., —CH₂C₆H₅, benzyl).

Representative aryl groups include C6-C12 aryl groups including, forexample, phenyl including substituted phenyl groups (e.g., benzoicacid).

X⁻ is the counter ion associated with the cationic center. The counterion can be the counter ion that results from the synthesis of thecationic polymers or the monomers (e.g., Cl⁻, Br⁻, I⁻). The counter ionthat is initially produced from the synthesis of the cationic center canalso be exchanged with other suitable counter ions to provide polymershaving controllable hydrolysis properties and other biologicalproperties. Representative hydrophobic counter ions includecarboxylates, such as benzoic acid and fatty acid anions (e.g.,CH₃(CH₂)_(n)CO₂ ⁻ where n=1-19); alkyl sulfonates (e.g., CH₃(CH₂)_(n)SO₃⁻ where n=1-19); salicylate; lactate; bis(trifluoromethylsulfonyl)amideanion (N⁻(SO₂CF₃)₂); and derivatives thereof. Other counter ions alsocan be chosen from chloride, bromide, iodide, sulfate; nitrate;perchlorate (ClO₄); tetrafluoroborate (BF₄); hexafluorophosphate (PF₆);trifluoromethylsulfonate (SO₃CF₃); and derivatives thereof. Othersuitable counter ions include hydrophobic counter ions and counter ionshaving therapeutic activity (e.g., an antimicrobial agent, such assalicylic acid (2-hydroxybenzoic acid), benzoate, lactate.

For the monomers, R₁ and R₂ is selected from hydrogen, fluoride,trifluoromethyl, and C1-C6 alkyl (e.g., methyl, ethyl, propyl, butyl).In one embodiment, R₁, R₂, and R₄ are hydrogen. In one embodiment, R₁,R₂, and R₄ are methyl.

Representative zwitterionic star polymers of the invention areillustrated in FIGS. 1 and 2.

FIG. 1 is a schematic illustration of the synthesis of a representativestar polymer of the invention having zwitterionic (polycarboxybetaine)branches. In this star polymer the branches are shown terminating with abromomethyl group, which is the radical initiator group for continuedpolymerization (e.g., atom transfer radical polymerization, ATRP). Thestar polymer was prepared by ATRP from the tetrafunctional core havingthe illustrated radical initiator groups (—O(C═O)—C(CH₃)₂—Br) andcarboxybetaine methacrylate (CH₂═C(CH₃)—C(═O)—CH₂CH₂—N(CH₃)₂⁺—CH₂CH₂—CO₂ ⁻, CBMA) monomers.

FIG. 2 is a schematic illustration of the synthesis of a representativestar polymer of the invention having zwitterionic (polycarboxybetaine)branches terminated with a thiol group (—CH₂—SH). This star polymer wasprepared from the star polymer illustrated in FIG. 1 by click chemistry(reaction of the star polymer with sodium azide and 3-thioacetylene).

Representative functional groups for the zwitterionic and mixed chargepolymers of the invention include OH, NH, NH₂, SH, N₃, CH═CH₂, C≡CH,COOH, CHO, imidoester, haloacetyl, hydrazide, alkoxyamine, aryl azide,diazirine, maleimide, carbodiimide, N-hydroxysuccinimide (NHS),thiazolidine-2-thione, pyridyldisulfide, difluorinatedcyclooctyne,Staudinger reagent pairs, isocyanate, isothiocyanate, thioether,sulfhydryl, hydrazine, hydroxymethyl phosphine, sulfo-NHS ester,pentafluorophenyl ester, sulfonylazide, 5H-dibenz[b,f]azepine, and theirderivatives.

In certain embodiments, the functionalized zwitterionic polymer hasformula (A):

wherein

(C) is a core (e.g., C1-C50 alkylene, arylene, acrylate, amine, amide,or other macromolecular cores);

R₁, R₄, R₅, R₈, and M are independently selected from the groupconsisting of C—C6 alkylene and C6-C12 arylene.

R₂ and R₃ are independently selected from the group consisting of C1-C6alkylene, C6-C12 arylene, —O(CH₂)_(m)—, —S(CH₂)_(m)—, —C(═O)(CH₂)_(m)—,—C(═S)(CH₂)_(m)—, —C(═NH)(CH₂)_(m)— and —NH(CH₂)_(m)—, —(CH₂)_(m)C(═O)—,—(CH₂)_(m)OC(═O)—, —(CH₂)_(m)NHC(═O)—, —(CH₂)_(m)NHC(═S)—,—(CH₂)_(m)SC(═O)—, —(CH₂)_(m)SC(═S)—, —(CH₂)_(m)NHC(═NH)—, and—(CH₂)_(m)C(═O)NHNH—, wherein m is an integer from 1 to 20;

K is a cationic center selected from the group consisting of ammonium,imidazolium, triazolium, pyridinium, morpholinium, and other nitrogen-,sulfide-, phosphate-based cations, such as ammoniophosphinates,ammonio(alkoxy)dicyanoethenolates, ammonioboronates,sulfoniocarboxylates, and oxypyridinebetaines;

R₆ and R₇ are independently selected from the group consisting ofhydrogen and C1-C6 alkyl, or taken together with K form a cationiccenter selected from the group consisting of ammonium, imidazolium,triazolium, pyridinium, morpholinium, and other nitrogen-, sulfide-,phosphate-based cations, such as ammoniophosphinates,ammonio(alkoxy)dicyanoethenolates, ammonioboronates,sulfoniocarboxylates, and oxypyridinebetaines;

A₁ is C, SO, SO₂, P, or PO⁻;

n is an integer from 5 to about 10,000;

N_(c) is core multiplicity and is an integer from 1 to 1000; and

X is a functional group selected from the group consisting of OH, NH,NH₂, SH, N₃, CH═CH₂, C≡CH, COOH, CHO, imidoester, haloacetyl, hydrazide,alkoxyamine, aryl azide, diazirine, maleimide, carbodiimide,N-hydroxysuccinimide (NHS), thiazolidine-2-thione, pyridyldisulfide,difluorinatedcyclooctyne, one of a Staudinger reagent pair, isocyanate,isothiocyanate, thioether, sulfhydryl, hydrazine, hydroxymethylphosphine, sulfo-NHS ester, pentafluorophenyl ester, sulfonylazide,5H-dibenz[b,f]azepine, and their derivatives.

Core multiplicity (N_(c)) is the total number of branches in thepolymer. In certain embodiments, the number of branches is from 2 to500, 3 to 100, 3 to 10, 3 to 6.

In certain embodiments, X is a thiol, a disulfide, a maleimide, or oneof a click chemistry reactive pair.

In other embodiments, the functionalized zwitterionic polymer hasformula (B):

wherein

(C), R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, M, K, n, and N_(c), are asdescribed above for (A);

Q⁻ is a counter ion selected from Cl⁻, Br⁻, I⁻, SO₄ ²⁻, NO₃ ⁻, ClO₄ ⁻,BF₄ ⁻, PF₆ ⁻, N(SO₂CF₃)₂ ⁻, SO₃CF₃ ⁻, or RCOO⁻ (where R is a C1-C20alkyl group), or lactate, benzoate, salicylate, and derivatives; and

R₉ is a functional group selected from the group consisting of hydrogen,trifluoromethyl, C1-C6 alkyl, C6-C12 aryl, —(CH₂)_(n)OH, —(CH₂)_(n)NH₂,—(CH₂)_(n)SH, —(CH₂)_(n)N₃, —(CH₂)_(n)CH═CH₂, —(CH₂)_(n)C≡CH,—(CH₂)_(n)COOH, —(CH₂)_(n)CHO, where n is 1 to 6, imidoester,haloacetyl, hydrazide, alkoxyamine, aryl azide, diazirine, maleimide,carbodiimide, N-hydroxysuccinimide (NHS), thiazolidine-2-thione,pyridyldisulfide, difluorinatedcyclooctyne, one of a Staudinger reagentpair, isocyanate, isothiocyanate, thioether, sulfhydryl, hydrazine,hydroxymethyl phosphine, sulfo-NHS ester, pentafluorophenyl ester,sulfonylazide, 5H-dibenz[b,f]azepine, and their derivatives.

In further embodiments, the functionalized zwitterionic polymer hasformula (C):

wherein

(C), R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, M, K, Q, X, n, and N_(c), areas described above for (A) and (B).

In other embodiments, the functionalized zwitterionic polymer hasformula (D):

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, Q, A₁, N_(c), and X are asdescribed above for (A), (B), and (C),

M₁, M₂, and M₃ are independently selected and are as described above forM for (A), (B), and (C);

K₁ and K₂ are independently selected and are as described above for Kfor (A), (B), and (C);

P₂ and P₃ are as described above for R₂ and R₃;

P₄, P₅, and P₈ are as described above for R₄, R₅, and R₈, respectively;

P₆ and P₇ are as described above for R₆ and R₇;

Q₂ and Q₃ as described above for R₂ and R₃;

Q₄ and Q₅ are as described above for R₄ and R₅;

R₉, F, and X are functional groups independently selected from the groupconsisting of —(CH₂)_(n)OH, —(CH₂)_(n)NH₂, —(CH₂)_(n)SH, —(CH₂)_(n)N₃,—(CH₂)_(n)CH═CH₂, —(CH₂)_(n)C≡CH, —(CH₂)_(n)COOH, —(CH₂)_(n)CHO, where nis 1 to 6, imidoester, haloacetyl, hydrazide, alkoxyamine, aryl azide,diazirine, maleimide, carbodiimide, N-hydroxysuccinimide (NHS),thiazolidine-2-thione, pyridyldisulfide, difluorinatedcyclooctyne, oneof a Staudinger reagent pair, isocyanate, isothiocyanate, thioether,sulfhydryl, hydrazine, hydroxymethyl phosphine, sulfo-NHS ester,pentafluorophenyl ester, sulfonylazide, 5H-dibenz[b,f]azepine, and theirderivatives.

m is an integer from 5 to about 10,000;

n is an integer from 5 to about 10,000; and

q is an integer from 5 to about 10,000.

In certain embodiments, the functionalized mixed charge copolymer hasformula (E):

wherein

(C), R₁, R₂, R₃, R₅, R₆, R₇, M, K, A₁, n, N_(c), and X are as describedabove for (A), and

N is as described above for M;

R₈ is as described above for R₆ and R₇.

R₉ is as described above for R₂ and R₃.

R₁₀ is as described above for R₅.

In other embodiments, the functionalized mixed charge copolymer hasformula (F):

wherein

(C), R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, M, K, N, A₁, n, and N_(c),are as described above for (E), Q⁻ is as described above for (B), andR₁₁ is selected from the group consisting of —(CH₂)_(n)OH,—(CH₂)_(n)NH₂, —(CH₂)_(n)SH, —(CH₂)_(n)N₃, —(CH₂)_(n)CH═CH₂,—(CH₂)_(n)C≡CH, —(CH₂)_(n)COOH, —(CH₂)_(n)CHO, where n is 1 to 6,imidoester, haloacetyl, hydrazide, alkoxyamine, aryl azide, diazirine,maleimide, carbodiimide, N-hydroxysuccinimide (NHS),thiazolidine-2-thione, pyridyldisulfide, difluorinatedcyclooctyne, oneof a Staudinger reagent pair, isocyanate, isothiocyanate, thioether,sulfhydryl, hydrazine, hydroxymethyl phosphine, sulfo-NHS ester,pentafluorophenyl ester, sulfonylazide, 5H-dibenz[b,f]azepine, and theirderivatives.

In further embodiments, the functionalized mixed charge copolymer hasformula (G):

wherein

(C), R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, M, K, N, A₁, n,N_(c), and X are as described above for (E) and (F).

In other embodiments, the functionalized mixed charge copolymer hasformula (H):

wherein

(C), R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, A₁, N_(c), Q⁻, and Xare as described above for (G);

M₁, M₂, and M₃ are independently M as described above for (G);

N₁ and N₂ are independently N as described above for (G);

K₁ and K₂ are independently K as described above for (G);

P₂, P₃, and P₉ are as for R₂, R₃, and R₉, respectively;

P₄ is as for R₄;

P₅ and P₁₀ are as for R₅ and R₁₀, respectively;

P₆, P₇, and P₈ are as for R₆, R₇, and R₈, respectively;

Q₂, Q₃, and Q₄ are as for R₂, R₃, and R₄, respectively;

F is as described above for (D); and

m, n, and q are as described above for (D).

Zwitterionic and Mixed Charge Polymeric Hydrogels

In a further aspect, the invention provides hydrogels prepared from thefunctionalized zwitterionic and mixed charge star polymers.Functionalized zwitterionic and mixed charge polymers and copolymers canbe used to form homopolymers or copolymers (e.g., hydrogels) by use ofthe same polymer (e.g., via disulfide linkage of thiol terminatedpolymers), two or more polymers (e.g. via click chemistry), or withother multifunctional molecules, oligomers, and polymers.

Poly(carboxybetaine) (pCB) polymers are highly functionalizable due tothe presence of abundant carboxylic acid (COOH) groups in the polymer(i.e., present in each constitutional unit). Such functionality rendersthe polymer versatile for further functionalized with simplechemistries. One such chemistry is the coupling of the COOH group withamine functional compounds. This chemistry is simple to carry out due tothe high efficiency of coupling agents (e.g.,N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride/N-hydroxysuccinimide (EDC/NHS)). Coupling chemistry can beused to functionalize pCB polymers with a variety of different amines.When amines are selected that contain complimentary groups for clickchemistry, pairs of click-reactive pCB polymers can be obtained.Hydrogel of the invention can be prepared by simply mixing the twopolymers with complimentary functional groups. In other embodiments, asuitable bifunctional crosslinker or other complimentary functionalizedcompound can be mixed with a reactive pCB polymer to provide a hydrogel.These concepts are illustrated in FIGS. 9-11.

In one embodiment, first and second zwitterionic polymers (e.g., pCBs)functionalized with complimentary clickable groups (R and R*) arecombined to provide a hydrogel as shown in Scheme 1.

Referring to FIG. 9, a first polymer bearing pendant first reactivegroups (R) and a second polymer bearing pendant second reactive groups(R*) are combined to provide a representative hydrogel of the invention.The first and second polymers can be prepared from the same polymer asshown in FIG. 9 (e.g., a zwitterionic polymer or mixed charge copolymerof the invention). The hydrogel is formed by crosslinking the first andsecond polymers through the complimentary first and second reactivegroups (e.g., complimentary first and second click groups)

In another embodiment, first and second complimentary reactive (e.g.,click-reactive) polymers can be prepared by copolymerization ofzwitterionic monomer (e.g., CBMA) and first and second comonomers,respectively. The first comonomer includes a first reactive group (e.g.,a zwitterionic monomer that further includes the first reactive group,such as a click-reactive group). The second comonomer includes a secondreactive group (e.g., a zwitterionic monomer that further includes thesecond reactive group, such as a click-reactive group). Thiscopolymerization strategy may be used where a high degree offunctionalization is required, such as for preparing a high-strengthinjectable hydrogel to mimic cartilage or other tissue. By thesemethods, functionality and overall zwitterionic character of the polymeris maintained. This strategy is shown in FIG. 10.

Referring to FIG. 10 path 2 a, a first zwitterionic copolymer isprepared by copolymerization of a zwitterionic monomer (denoted CBmonomer) (e.g., CBMA) and a first comonomer (denoted CB-click monomer)that includes a first reactive group (i.e., a zwitterionic comonomerthat further includes the first reactive (e.g., first click-reactive)group). Referring to FIG. 10 path 2 b, a second zwitterionic copolymeris prepared by copolymerization of a zwitterionic monomer (denoted CBmonomer) (e.g., CBMA) and a second comonomer (denoted CB-click monomer)that includes a second reactive group (e.g., a zwitterionic monomer thatfurther includes the second reactive (e.g., second click-reactive)group, as shown in 2 a. The hydrogel is formed by crosslinking the firstand second polymers through the complimentary first and second reactivegroups (e.g., complimentary first and second click reactive groups).

In a further embodiment, zwitterionic polymers may be end-groupfunctionalized with complimentary reactive (e.g., click-reactive)groups. See FIG. 11. These end-group functionalized polymers can be usedwith the polymers and copolymers illustrated in FIGS. 9 and 10. Thepreparation of end-group functionalized polymers and the hydrogel formedby their mixing is shown in FIG. 11.

Referring to FIG. 11 path 3 a, a first zwitterionic polymer is end-groupfunctionalized with a first reactive group (e.g., first click-reactivegroup). Referring to FIG. 11 path 3 b, a second zwitterionic polymer isend-group functionalized with a second reactive group (e.g., secondclick-reactive group). The hydrogel is formed by crosslinking the firstand second polymers through the complimentary first and second reactivegroups (e.g., complimentary first and second click reactive groups).

The crosslinking (hydrogel-forming) chemistries above are described asreaction between complimentary reactive groups (i.e., first and secondreactive groups). It will be appreciated that the complimentary reactivegroups can be any pair of reactive groups having reactivity suitable forbond formation, preferably reactivity suitable for bond formation attemperatures between room and physiological temperature. In certainembodiments, the complimentary reactive groups are click groups(click-reactive pairs), which are well known in the art.

Representative zwitterionic click hydrogels formed using the strategiesdescribed above are set forth in Examples 1-6.

The hydrogels can take the form of a scaffold, an injectable hydrogel ornanogel with or without biotic compounds. In certain embodiments, thehydrogels of the present invention further comprise encapsulated smallmolecules, nanomaterials, nucleic acids, polysaccharides, proteins, andcells. Such hydrogels have applications in medical and pharmaceuticalsensing, orthopedics, cardiovascular, internal ophthalmic, aesthetic,ocular and drug/protein/drug delivery. The hydrogels can be attached tosurfaces for medical and engineering applications. In addition toforming hydrogels, functionalized zwitterionic polymers can also beattached to biotic or non-biotic molecules and substrates. Furthermore,for functionalizable polycarboxybetaine (PCB) polymers, the polymer canbe conjugated directly in a degradable or non-degradable way whilebranches can be replaced by as acrylamide, oxazoline, andvinylpyrrolidone.

The hydrogels of the invention can be used for objects, devices, andcomponents, such as implantable biosensors; wound care devices, gluesand sealants, a contact lens; a dental implant; an orthopedic devicesuch as an artificial joint, an artificial bone, an artificialligaments, and an artificial tendon; a cardiovascular device such as acathether, an artificial valve, an artificial vessel, an artificialstent, LVADs, or a rhythm management device; gastroenterology devicessuch as feeding tubes, alimentary canal clips, gastro-intestinalsleeves, or gastric balloons; OB/Gyn devices such as implantable birthcontrol devices or vaginal slings; nephrology devices such asanastomotic connectors or subdermal ports; neurosurgery devices such asnerve guidance tubes, cerebrospinal fluid drains or shunts, dermatologydevices such as skin repair devices an ophthalmic device such as ashunt, otorhinolaryngology devices such as stents, cochlear implants,tubes, shunts or spreaders, an intra-ocular lense; an aesthetic implantsuch as a breast implant, a nasal implant, and a cheek implant; aneurologic implant such as a nerve stimulation device, a cochlearimplant, and a nerve conduit; a hormone control implant such as a bloodsugar sensor and an insulin pump; an implanted biosensor; an access portdevice; and a tissue scaffold pulmonic devices such as valves formanagement of COPD or artificial lungs; radiology devices such asradio-opaque or sono-opaque markers; or urology devices such ascatheters or artificial urethrae.

The following is a description of representative hydrogels of theinvention and their use for cell culture and tissue scaffolds.

Functionalized Zwitterionic Polymers-Based Hydrogel for IsletsEncapsulation.

Functionalized star-shaped poly(carboxybetaine) polymers (pCB) preparedby polymerization of CBMA were synthesized via a combination of ATRP andclick chemistry. Thiol group-terminated pCB polymers was synthesized,and pCB hydrogels were formed by mixing thiol-terminated pCBs with1,2-bis(maleimido)ethane together under physiological conditions.

The synthesis of star pCB polymer via ATRP was conducted as illustratedin FIG. 1. Briefly, CBMA, 2,2′-bipyridine (bpy), catalysts, andtetrafunctional initiator, pentaerythritoltetrakis(2-bromoisobutyrate)were placed in a 10 mL reaction tube, and the mixture was subjected tothree freeze-pump-thaw cycles. The mixture was held at room temperaturefor 20 min, and water and methanol were added at a 1:1 ratio. Thereaction was allowed to continue at room temperature under stirring for8 h. The polymer product was recovered after treatment with alumina, andfinally purified by precipitation twice into acetone. The molecularweight of the polymer can be tuned by the stoichiometric ratio betweenthe monomer and the initiator. Star polymers with molecular weights of5000, 20,000 and 50,000 were synthesized. Other living polymerizationmethods, such as RAFT. can also be applied to synthesize the polymer.The functionalized star pCB polymers were prepared by click chemistry.As shown in FIG. 2, after purification, the terminal bromine groups ofthe initially formed star pCB were transformed into azido groups by anucleophilic substitution reaction with sodium azide in water. Theproduct was purified by dialysis. Lyophilization was used to remove thewater. pCB-N₃ (pCB-azide) chains were then reacted with the alkyne-SH inmethanol with CuBr/PMDETA as catalyst to produce the star pCB polymerswith four arm. As illustrated in FIG. 2, thiol-terminated star pCB wasobtained after purification via dialysis and lyophilization.

FIGS. 3A and 3B schematically illustrate the preparation of arepresentative hydrogel of the invention prepared from suitablyfunctionalized star polymers of the invention. FIG. 3A illustrates thesyntheses of two representative star polymers of the invention: (a) astar polymer having zwitterionic (polycarboxybetaine) branchesterminated with a thiol group (—CH₂—SH) denoted pCB-A; and (b) a starpolymer having zwitterionic (polycarboxybetaine) branches terminatedwith a pyridyl disulfide group (—CH₂—S—S—C₅H₄N) denoted pCB-B. Thesestar polymers were prepared from the star polymer illustrated in FIG. 1by click chemistry (reaction of the star polymer with sodium azide and(a) 3-thioacetylene or (b) 3-pyridyl disulfide acetylene). FIG. 3Billustrates the preparation of representative hydrogels of the inventionprepared from reaction of the star polymers (pCB-A and pCB-B)illustrated in FIG. 3A. The hydrogels can be formed by simply mixingpCB-A with pCB-B in the presence of cells.

In vitro islet encapsulation and analysis in pCB hydrogel was evaluated.Rat islets were mixed with 5% thiol-terminated pCB-A (MW=15000) inRPMI-1640 medium. Then, 1,2-bis(maleimido)ethane was added to themixture and the system was allowed to cure at 37° C. for 5 min.Hydrogel-encapsulated islets and control islets were cultured withRPMI-1640 culture medium, supplemented with 10% FBS at 37° C., withmedia changed every two days. As shown in FIG. 4A, encapsulated isletsexhibited very high viability after the 18-day culture in vitro. Glucoseconsumption in culture media was used to measure the metabolic activityof hydrogel-encapsulated islets maintained in culture. Glucosemetabolism was measured primarily by the glucose utilization in mediumby the cultured islets and shown as an indicator not only for isletviability, but also cellular activity. The uptake of glucose in mediumwas measured every 48 h over the 18 day period to assess islet quality.Glucose uptake in encapsulated islets was maintained near 100% of theday 1 level until day 18 (FIG. 4B). In control islets, glucoseconsumption decreased to 72% by day 7, and further declined as timeprogressed. By day 14 and 18, only 59% and 38% of the glucose wasutilized, respectively. Overall, encapsulated islets maintained muchhigher metabolic activity as compared to control islets (p<0.01). Toquantify the biosynthetic capacity of insulin, cultured islets wereincubated in either low (3 mM) or high (17 mM) glucose medium for 18 hon day 0 and day 18 (FIG. 4B). Hydrogel-encapsulated islets maintainedinsulin secretion levels of freshly isolated islets (day 0) in bothhigh- and low-glucose exposure (p=0.13-0.40). In contrast, the glucoseresponsiveness was not preserved in the control cultured islets testedon day 18 (p<0.05, vs. control on day 0 and encapsulated islets on day18). The decreased insulin storage/secretion ability of the controlislets was also shown by decreased insulin staining by dithizone (DTZ)on day 18 (FIG. 4C). To further assess beta cell function, dynamicglucose stimulated insulin release was tested in a perifusion systemusing islets that were cultured with or without hydrogel encapsulationfor 18 days. Although the same number of islets was present in both theencapsulated and control samples on day 0, islets in the control groupdisintegrated over time and did not show stimulated insulin secretion.In contrast, encapsulated islets responded well to glucose stimulation(FIG. 4D).

Hydrogels can also be formed by mixing two different polymers. Forexample, as presented in FIG. 3A, thiol-terminated pCB-A andpyridyldisulfide-terminated pCB-B were synthesized via a combination ofATRP and click chemistry. The hydrogel can be formed by mixing pCB-Awith pCB-B under physiological conditions (see FIG. 3B). Because thehydrogel can be formed under physiological conditions, cells such asstem cells, immune cells, neural cells, hormone-secreting cells, andheart cells can be added in the process of gelation in order to beencapsulated within the hydrogels. In addition, due to the hydrogel isbeing crosslinked with disulfide bonds, the hydrogel can be degraded bybiological agents such as cysteine.

Functionalized Zwitterionic Polymers-Based Hydrogel for Peripheral BloodStem Cell (PBSC) Cell Encapsulation.

As described above for rat islet cells, peripheral blood stem cell(PBSC) cell were also encapsulated in a representative hydrogel of theinvention.

FIGS. 5A and 5B present data for encapsulated peripheral blood stem cell(PBSC) cells cultured in a representative hydrogel (pCB hydrogel) of theinvention. FIG. 5A illustrates hydrogel modulus over time (hydrogeldegradation). FIG. 5B is an image showing the results of a LIVE/DEADassay performed on encapsulated PBSC cells within the pCB hydrogelsafter 7-day in vitro culture.

The hydrogels of the invention can further include components oradditives that are advantageously administered by the methods of theinvention.

In certain embodiments, the hydrogel further includes cells. In theseembodiments, the cells are contained within or encapsulated in thehydrogel. The nature of the cell encapsulated in the hydrogel is notlimited (natural cells and genetically modified cells, as well astriturates thereof). Representative cells that are advantageouslyencapsulated in the hydrogel include exocrine secretory epithelialcells, hormone secreting cells, keratinizing epithelial cells, wetstratified barrier epithelial cells, sensory transducer cells, autonomicneuron cells, sense organ and peripheral neuron supporting cells,neurons and glial cells, lens cells, hepatocytes, adipocytes, lipocytes,barrier function cells, kidney cells, heart cells, extracellular matrixcells, contractile cells, blood and immune system cells such aserythrocytes, monocytes, neutrophils, mast cells, T cells, stem cells,germ cells, nurse cells, interstitial cells, progenitor cells, andhematopoietic stem cells.

In certain embodiments, the hydrogel further includes viruses. In theseembodiments, the viruses are contained within or encapsulated in thehydrogel. Representative viruses include retroviruses and adenoviruses.

In certain embodiments, the hydrogel further includes bacteria. In theseembodiments, the bacteria are contained within or encapsulated in thehydrogel. Representative bacteria include clostridial species, S.typhimurium, and E. coli.

Other materials advantageously encapsulated in the hydrogels includeproteins, peptides, nucleic acids (oligonucleotides), polysaccharides,small molecules (i.e., organic, inorganic and organometallic compoundshaving a molecular weight less than about 800 g/mole) and nanomaterials.Representative proteins include antibodies, antibody fragments, enzymes(e.g., therapeutic and protective enzymes), and peptides. Representativenucleic acids include DNAs (e.g., cDNA) and RNAs (e.g., siRNA, mRNA)Representative small molecules include thereapuetic agents anddiagnostic agents (e.g., fluorescent and magnetic resonance imagingagetns). Representative nanomaterials include carbon nanostructures(e.g., carbon nanotubes and graphenes) and quantum dots.

Other materials advantageously encapsulated in the hydrogels includelipoplexes, polymerosomes, polyplexes, dendrimers, and inorganicparticles.

In certain embodiments, the hydrogel is crosslinked with a degradablecrosslinker. Representative degradable crosslinkers include peptides,polysacharrides, anhydride crosslinkers, dissulfide crosslinkers, andpolyester crosslinkers. In these embodiments, the hydrogel can bedesigned to degrade under specific circumstances (e.g., physiologicalconditions), such as by hydrolysis or digestion by enzymes.

In certain embodiments, the hydrogel of the invention is prepare fromfunctionalized zwitterionic polymers and has formula (J):

wherein

(C1) and (C2) are cores and are independently selected from acrylatefunctionalized C1-C6 alkyl, and C6-C12 aryl groups; amine functionalizedC1-C6 alkyl, and C6-C12 aryl groups; amide functionalized C1-C6 alkyl,and C6-C12 aryl groups; epoxy functionalized C1-C6 alkyl, and C6-C12aryl groups; C1-C6 alkyl, and C6-C12 aryl groups; or othermacromolecular core;

Y₁ and Y₂ are linking groups and independently selected from—O(CH₂)_(n)—, —S(CH₂)_(n)—, —C(═O)(CH₂)_(n)—, —C(═S)(CH₂)_(n)—,—C(═NH)(CH₂)_(n)—, —NH(CH₂)_(n)—, —(CH₂)_(n)C(═O)—, —(CH₂)_(n)OC(═O)—,—(CH₂)_(n)NHC(═O)—, —(CH₂)_(n)NHC(═S)—, —(CH₂)_(n)—C(═O)—,—(CH₂)_(n)SC(═S)—, —(CH₂)_(n)NH—C(═NH)—, —(CH₂)C(═O)NHNH—, wherein n isan integer from 1 to 20, and other linkages which can be formed fromfunctional groups X defined above;

R₁-R₈ are as described above for (A);

R₉-R₁₆ are as for R₁-R₈; respectively,

M is as described above for (A);

N is M;

K₁ and K₂ are independently as described above for K for (A);

A₁ is C, SO, SO₂, P, or PO⁻;

A₂ is C, SO, SO₂, P, or PO⁻;

m is an integer from 5 to about 10,000;

n is an integer from 5 to about 10,000;

N_(c1) and N_(c2) are core multiplicities and are integers independentlyselected from 2 to 1000; and

q is the an integer intermediate N_(c1) and N_(c2).

In other embodiments, the hydrogel of the invention is prepare fromfunctionalized zwitterionic polymers and has formula (K):

wherein the definitions of the substituents are as described above for(J), P⁻ is as described for Q⁻ for (B), and R₁₇ and R₁₈ are functionalgroups and are independently selected from the groups defined by R₉ for(B).

In certain embodiments, the hydrogel of the invention is prepare fromfunctionalized mixed charge copolymers and has formula (L):

wherein

(C1) and (C2) are cores and are independently selected from acrylatefunctionalized C1-C6 alkyl, and C6-C12 aryl groups; amine functionalizedC1-C6 alkyl, and C6-C12 aryl groups; amide functionalized C1-C6 alkyl,and C6-C12 aryl groups; epoxy functionalized C1-C6 alkyl, and C6-C12aryl groups; C1-C6 alkyl, and C6-C12 aryl groups; or othermacromolecular core;

Y₁ and Y₂ are linking groups and independently selected from—O(CH₂)_(n)—, —S(CH₂)_(n)—, —C(═O)(CH₂)_(n)—, —C(═S)(CH₂)_(n)—,—C(═NH)(CH₂)_(n)—, —NH(CH₂)_(n)—, —(CH₂)_(n)C(═O)—, —(CH₂)_(n)OC(═O)—,—(CH₂)_(n)NHC(═O)—, —(CH₂)_(n)NHC(═S)—, —(CH₂)_(n)—C(═O)—,—(CH₂)_(n)SC(═S)—, —(CH₂)_(n)NH—C(═NH)—, —(CH₂)C(═O)NHNH—, wherein n isan integer from 1 to 20, and other linkages which can be formed fromfunctional groups X defined above;

R₁-R₁₀ are as described above for (E);

R₁₁-R₂₀ are as for counterpart R₁-R₁₀;

M₁ and M₂ are independently as described above for M for (K);

N₁ and N₂ are independently as described above for N for (K);

K₁ and K₂ are independently as described above for K for (K);

A₁ is C, SO, SO₂, P, or PO⁻;

A₂ is C, SO, SO₂, P, or PO⁻;

m is an integer from 5 to about 10,000;

n is an integer from 5 to about 10,000;

N_(c1) and N_(c2) are core multiplicities and are integers independentlyselected from 2 to 1000; and

q is the an integer intermediate N_(c1) and N_(c2).

In other embodiments, the hydrogel of the invention is prepare fromfunctionalized mixed charge copolymers and has formula (M):

wherein the definitions of the substituents are as described above for(L), P⁻ is as for Q⁻ as described for (F), and R₂₁ and R₂₂ arefunctional groups and are independently selected from the groups definedby R₈ for (F).

Surfaces Treated with Zwitterionic or Mixed Charge Polymeric Hydrogels

In another aspect, the invention provides surfaces that have beentreated with the zwitterionic or mixed charge hydrogels of theinvention. The hydrogels of the invention, which in certain embodimentsare hydrolyzable to zwitterionic polymers or mixed charge copolymers,can be advantageously used as coatings for the surfaces of a variety ofdevices including, for example, medical devices.

The hydrogels of the invention are advantageously used to coat surfacesto provide biocompatible, antimicrobial, and nonfouling surfaces.Accordingly, in another aspect, the invention provides devices andmaterials having a surface (i.e., one or more surfaces) to which havebeen applied (e.g., coated, covalently coupled, ionically associated,hydrophobically associated) one or more crosslinked zwitterionichydrogels of the invention. Representative devices and carriers that maybe advantageously treated with a hydrogel of the invention, modified toinclude a hydrogel of the invention, or incorporates a hydrogel of theinvention include:

particle (e.g., nanoparticle) having a surface treated with, modified toinclude, or incorporates a hydrogel of the invention;

drug carrier having a surface treated with, modified to include, orincorporates a material of the invention;

non-viral gene delivery system having a surface treated with, modifiedto include, or incorporates a hydrogel of the invention;

biosensor having a surface treated with, modified to include, orincorporates a hydrogel of the invention;

devices for bioprocesses or bioseparations, such as membranes formicrobial suspension, hormone separation, protein fractionation, cellseparation, waste water treatment, oligosaccharide bioreactors, proteinultrafiltration, and diary processing having a surface treated with,modified to include, or incorporates a hydrogel of the invention;

implantable sensor having a surface treated with, modified to include,or incorporates a hydrogel of the invention;

subcutaneous sensor having a surface treated with, modified to include,or incorporates by a hydrogel of the invention;

implant, such as a breast implant, cochlear implant, and dental implanthaving a surface treated with, modified to include, or incorporates ahydrogel of the invention;

contact lens having a surface treated with, modified to include, orincorporates a hydrogel of the invention;

tissue scaffold having a surface treated with, modified to include, orincorporates a hydrogel of the invention;

implantable medical devices, such as an artificial joint, artificialheart valve, artificial blood vessel, pacemaker, left ventricular assistdevice (LVAD), artery graft, and stent having a surface treated with,modified to include, or incorporates a hydrogel of the invention; and

medical devices, such as an ear drainage tube, feeding tube, glaucomadrainage tube, hydrocephalous shunt, keratoprosthesis, nerve guidancetube, urinary catheter, tissue adhesive, and x-ray guide having asurface treated with, modified to include, or incorporates by a hydrogelof the invention.

Other representative substrates and surfaces that may be advantageouslytreated with a hydrogel of the invention, modified to include a hydrogelof the invention, or incorporates a hydrogel of the invention includefabrics and such as in clothing (e.g., coats, shirts, pants,undergarments, including such as worn by hospital and militarypersonnel), bedding (e.g., blankets, sheets, pillow cases, mattresses,and pillows), toweling, and wipes.

Other representative substrates and surfaces that may be advantageouslytreated with a hydrogel of the invention, modified to include a hydrogelof the invention, or incorporates a hydrogel of the invention includeworking surfaces such as tabletops, desks, and countertops.

The hydrogels of the invention can be coated onto a variety of surfacesincluding surfaces of objects, devices, and components such asimplantable biosensors; wound care devices, glues and selants, a contactlens; a dental implant; an orthopedic device such as an artificialjoint, an artificial bone, an artificial ligaments, and an artificialtendon; a cardiovascular device such as a cathether, an artificialvalve, an artificial vessel, an artificial stent, LVADs, or and a rhythmmanagement device; gastroenterology devices such as feeding tubes,alimentary canal clips, gastro-intestinal sleeves, or gastric balloons;OB/Gyn devices such as implantable birth control devices or vaginalslings; nephrology devices such as anastomotic connectors or subdermalports; neurosurgery devices such as nerve guidance tubes, cerebrospinalfluid drains or shunts, dermatology devices such as skin repair devicesan ophthalmic device such as a shunt, otorhinolaryngology devices suchas stents, cochlear implants, tubes, shunts or spreaders, anintra-ocular lense; an aesthetic implant such as a breast implant, anasal implant, and a cheek implant; a neurologic implant such as a nervestimulation device, a cochlear implant, and a nerve conduit; a hormonecontrol implant such as a blood sugar sensor and an insulin pump; animplanted biosensor; an access port device; and a tissue scaffoldpulmonic devices such as valves for management of COPD or artificiallungs; radiology devices such as radio-opaque or sono-opaque markers; orurology devices such as catheters or artificial urethrae.

In Situ Zwitterionic and Mixed Charge Polymeric Hydrogels

In a further aspect, the invention provides a method for making azwitterionic or mixed charge polymeric hydrogel. In certain embodiments,the method is an in situ method, such as an in vivo method, in which asuitably functionalized zwitterionic star polymer or a suitablyfunctionalized mixed charge star copolymer is introduced to anenvironment where the hydrogel is desirably located. By virtue of thecomplimentary functional groups of the functionalized zwitterionic starpolymer or the functionalized mixed charge star copolymer, the polymersor copolymers are crosslinked to provide the hydrogel in situ.

When the environment is an in vivo location, the functionalizedzwitterionic star polymer or a functionalized mixed charge starcopolymer can be introduced to the desired site by, for example,injection.

By this method the hydrogel and its added cargo can be advantageouslyformed at desired in vivo sites.

In certain embodiments, two suitable functionalized zwitterionicpolymers of the invention, each having a functional group of a reactivepair (e.g., the first polymer bearing first functional groups and thesecond polymer bearing second functional groups, the first and secondfunctional groups being reactive toward one another to covalently couplethe first polymer to the second polymer), are administered such that thetwo polymers react to form a hydrogel when combined or come into contactwith each other in situ (e.g., in vivo after injection at the desiredsite).

In other embodiments, two functionalized mixed charged copolymers of theinvention, each having one functional group of a reactive pair (e.g.,the first polymer bearing first functional groups and the second polymerbearing second functional groups, the first and second functional groupsbeing reactive toward one another to covalently couple the first polymerto the second polymer), are administered such that the two polymersreact to form a hydrogel when combined or come into contact with eachother in situ (e.g., in vivo after injection at the desired site).

Zwitterionic and Mixed Charge Star Polymer Therapeutic Agent Conjugates

In another aspect, the invention provides zwitterionic and mixed chargepolymers and copolymers having therapeutic agents covalently coupledthereto. These conjugates are useful for therapeutic agent delivery.Controlled release of therapeutic agents from the polymers of theinvention by hydrolysis

The following is a description of a representative conjugate of theinvention and release of therapeutic agent from the conjugate.

FIG. 6 is a schematic illustration of the release of a representativetherapeutic agent from a representative star polymer of the inventionhaving zwitterionic (polycarboxybetaine) branches. The star polymer wasprepared by ATRP from the tetrafunctional core having the radicalinitiator groups and carboxybetaine methacrylate-therapeutic agentmonomers (CH₂═C(CH₃)—C(═O)—CH₂CH₂—N(CH₃)₂ ⁺—CH₂CH₂—CO₂-therapeuticagent). In the representative star polymer shown, the branches arefurther extended by extension polymerization with CBMA to providebranches having constitutional units that are zwitterionic in additionto constitutional units bearing the therapeutic agent. The star polymerillustrated in FIG. 6 is a block copolymer. It will be appreciated thatin certain embodiments, the star polymer is a random copolymer preparedby copolymerization of zwitterionic monomers (e.g., CBMA) andzwitterionic-therapeutic agent monomers (e.g., CBMA-therapeutic agent).On hydrolysis of the star polymer, the therapeutic agent is released.Hydrolysis of the star polymer and release of the therapeutic agentregenerates a zwitterionic constitutional unit in the star polymer. Asshown in FIG. 6, the therapeutic agent is ibuprophen, and the monomerfor synthesizing the star polymer capable of releasing ibuprophen isprepared by condensation of CBMA with ibuprophen.

As shown in FIG. 6, ibuprofen was incorporated into carboxybetainemethacrylate (CBMA) via anhydride formation reaction by stirring withdicyclohexylcarbodiimide (DCC) in dichloromethane (DCM) to provide apolymerizable monomer. A 4-arm star zwitterionic polymer was preparedfrom the monomer to provide a polymer therapeutic agent conjugate. Asshown in FIG. 6, the therapeutic agent is released by hydrolysis. Theresulting polymer after the hydrolysis is converted to its counterpartzwitterionic polymer.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range (i.e.,denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value).

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The following examples are presented for the purpose of illustrating,not limiting the invention.

EXAMPLES Example 1 Representative Hydrogel: First and SecondZwitterionic Polymers Cyclooctyne/Azide Chemistry

In this example, a representative hydrogel of the invention is preparedfrom first and second zwitterionic polymers. The first polymer includespendant first reactive groups (i.e., cyclooctyne) and the second polymerincludes pendant second reactive groups (i.e., azide).

Mixing the two polymers without any external stimuli provides thehydrogel.

The formation of the first and second polycarboxybetaine polymers andthe hydrogel formed from the polymers is shown below.

The polycarboxybetaine (pCB) polymer is functionalized in two differentreactions with a cyclooctyne amine and an azido amine, respectively,using the known EDC/NHS coupling. The pCB polymers are first activatedby EDC/NHS chemistry and then the polymer is purified by precipitationin THF. The activated polymer is then treated with respective amines.The polymer is again purified by precipitation in THF. The purifiedpolymer is dried under vacuum. The degree of functionalization ischaracterized by ¹H and ¹³C NMR. These two functionalized pCB polymersare mixed and then undergo the click reaction to form the hydrogel. Theextent of functionalization and hence the extent of crosslinking can beeasily varied by changing the amount of reactive amines.

Example 2 Functionalized Zwitterionic Copolymers

In this example, the preparation of a representative functionalizedzwitterionic copolymer of the invention is described. The representativefunctionalized copolymer includes pendant groups (i.e.,N-hydroxysuccinimide groups) that allow for covalent coupling of furtheractivating groups (complimentary reactive groups) to providecrosslinkable polymers useful for forming hydrogels of the invention.

Carboxybetaine methacrylate monomer (CBMA) is functionalized withN-hydroxysuccinimide (NHS-CBMA). This monomer is copolymerized withcarboxybetaine methacrylate monomer (CBMA) (see FIG. 11). The resultantpolymer is a random polymer of CBMA and NHS-CBMA. The copolymerizationis shown below.

The feed ratio of the two monomers can be varied to providefunctionalized polymers having different degrees of functionalization.The activated polymer can be readily further functionalized withsuitable amines, facilitating steps 1 a and 1 b in FIG. 9.

An NHS activated carboxybetaine monomer is synthesized first. Thismonomer is polymerized along with carboxybetainemethacrylate atdifferent feed ratios. The polymer obtained is already activated. Thispolymer is purified by precipitating in THF to remove all the smallmolecular impurities (e.g., unreacted monomers, initiators) and thenvacuum dried. The polymer is characterized using ¹H and ¹³C NMR.

Example 3 Representative Hydrogel: First and Second ZwitterionicPolymers Thiol/Maleimide Chemistry

In this example, a representative hydrogel of the invention is preparedfrom first and second zwitterionic polymers. The first polymer includespendant first reactive groups (i.e., thiol) and the second polymerincludes pendant second reactive groups (i.e., maleimide).

The formation of the first and second polycarboxybetaine polymers isshown below.

The synthesis of a random copolymer of carboxybetaine methacrylate andmaleimide functionalized CBMA monomer is shown below.

The synthesis of a random copolymer of carboxybetaine methacrylate andethanethiol functionalized CBMA monomer is shown below.

Mixing the two polymers without any external stimuli provides thehydrogel.

A functionalized carboxybetaine monomer is synthesized first withcomplimentary clickable groups. This functionalized monomer is randompolymerized with CBMA monomer. As representative examples, maleimide andthiol functionalized CBMA monomers are synthesized using amines based onmaleimide and thiol. The polymer is purified by precipitation in THF toremove unreacted monomers. The composition of the polymer is confirmedby NMR (¹H and ¹³C). The amount of functionalized units in the polymeris varied by adjusting the feed ratios. Thus complimentary clickablepolymers are synthesized. These polymers are mixed at 5-10 wt % in waterto form the hydrogel.

Example 4 Representative Zwitterionic Copolymer: Thiol Pendant Group

In this example, a modified carboxybetaine monomer is synthesized bymodification of the ammonium moiety retaining the negative charge on thecarboxylate to provide a thiol-functionalized zwitterionic monomer.

Copolymerization of the CBMA and the modified CBMA monomers to provide afunctionalized zwitterionic copolymer is shown below.

Such a monomer has advantages for high crosslinking rates. A similarmonomer and copolymer can be obtained for the corresponding click groupcounterpart to complete the procedure shown in FIG. 10.

Example 5 Representative Zwitterionic Copolymer Hydrogels: IsletsEncapsulation

In this example, islets encapsulation using representative zwitterionicpolymers is described.

Functionalized star-shaped pCB polymers were synthesized via acombination of ATRP and ‘Click’ reaction. Maleimide and thiolgroup-terminated pCB polymers with different molecular weights weresynthesized. pCB hydrogels were formed by mixing maleimide-terminatedpCB (pCB-A) with thiol-terminated pCB (pCB-B) together underphysiological conditions. The mechanical properties, equilibrium watercontent (EWC) and pore sizes of the hydrogel can be tuned by adjustingthe molecular weight of the polymers and the amount of used polymers.

Synthesis of Star pCB Polymer Via ATRP.

CBMA, 2,2′-bipyridine (bpy), catalysts, tetrafunctional initiator,pentaerythritol tetrakis(2-bromoisobutyrate) were placed in a 10 mLreaction tube, and the mixture was subjected to three freeze-pump-thawcycles. The mixture stayed under room temperature for 20 min, and waterand methanol were added at a 1:1 ratio. The reaction was allowed tocontinue at room temperature under stirring for 8 h. The polymer productwas recovered after treatment with alumina, and finally purified byprecipitation twice into acetone. The molecular weight of the polymercan be tuned by the stoichiometric ratio between the monomer and theinitiator. Star polymers with weight average molecular weights of 5000,20000 and 50000 were synthesized. Other living polymerization methodsuch as reversible addition-fragmentation chain-transfer (RAFT) can alsobe applied to synthesize the polymer.

Synthesis of Functionalized Star pCB Polymers Via Click Chemistry.

After purification, the terminal bromine groups of the star pCB wastransformed into azido groups by a nucleophilic substitution reactionwith sodium azide in water. The product was purified by dialysis.Lyophilization was used to remove the water. pCB-N₃ chains were thenreacted with the alkyne-containing compounds in methanol withCuBr/PMDETA as catalyst to produce the star pCB polymers with 4 armnumbers. Thiol terminated star-shaped pCB (pCB-A) and maleimideterminated star-shaped pCB (pCB-B) were obtained after purification viadialysis and lyophilization.

Islets-Free Assessment of the Gelling Properties.

pCB gels were formed by adding pCB-A with various polymer weightpercentage solutions of pCB-B in PBS at 37° C. See, e.g., FIG. 3. Thehydrogels obtained were washed thoroughly with distilled water to removethe unreacted polymers. The EWCs of the hydrogels were measured at 37°C. using a gravimetric method. The temperature was controlled by athermostatic water bath with a precision of +0.1° C. The samples wereimmersed in 0.1 M PBS buffer solutions (pH 7.4) for at least 24 h andthen taken out, blotted with wet filter paper to remove water on thesurface, and weighed on a microbalance. After crosslinking, hydrogelswere allowed to freely swell in PBS for 24 hours.

In Vitro Islet Function Assessment in pCB Hydrogel.

The biocompatibility of pCB hydrogels was tested in vitro. The viabilityand the function of the encapsulated porcine islets within pCB hydrogelswere tested. In addition, the inhibitory effect of pCB hydrogels on theactivation of the complement system, lymphocytes, monocytes, plateletsand PMNs and cytokine production were tested.

Islet Viability Assessment.

The viability of porcine islets was quantitatively monitored byluminescence intensity. Hydrogel-encapsulated islets or control isletsisolated from pigs (100 islets/well) were cultured for up to 28 days andluminescence intensity was measured using the Xenogen image system(Caliper Life Sciences, Hopkinton, Mass.) by adding 0.75 mg/mLD-luciferin to the islet culture medium. Islet viability was alsoqualitatively assessed using Live/Dead staining at desired time points.Live/Dead staining was done by incubating cell-laden hydrogels in PBScontaining 0.24 mM fluorescein diacetate and 7.5 mM propidium iodide for10 min. Hydrogels were then rinsed in PBS and imaged by fluorescencemicroscopy (Olympus IX50, Olympus America, Central Valley, Pa.).

Glucose Consumption During Islet Culture.

Culture media was collected every 48 h during medium exchange, andmonitored for glucose level with a One Touch glucose meter (LifeScan,Milpitas, Calif.) in duplicate and averaged. Control medium (Ct) withoutislets were used as control for each time point. Differences (C0−I0) inthe medium glucose level with (I0) and without (C0) islets in the first48 h are set as baseline cell glucose consumption for each sample.Subsequent changes in glucose levels in samples with islets (It) arecalculated as: Percent glucose uptake=(Ct−It)*100/(C0−I0).

Long-Term Response to Glucose Stimulated Insulin and C-Peptide Release.

Islets cultured with or without hydrogel as described above werehand-picked after 14 days in culture and collected in micro-tubes(single islet/tube, sexplicate), washed with RPMI 1640 medium containing3 mM glucose (low-glucose media) supplemented with 5% FBS 3 times.Islets were cultured in a non-tissue culture coated 96 well plate witheither low-glucose or high-glucose (17 mM glucose) media for 16 h atwhich time concentrations of insulin and C-peptide in supernatant mediawere measured using Porcine Insulin and Porcine C-peptide Elisa kits(Mercodia, Winston Salem, N.C.) according to the manufacturer'sinstructions.

Short-Term Response to Glucose Stimulated Insulin Release by PerifusionAssay.

Islets were cultured with or without hydrogel as previously described(200 islets/sample on day 0). On day 28, islets were collected andsandwiched-trapped between a bi-layer of microbeads (Biorad, Hercules,Calif.) in a minicolumn and perifused at 37° C. with Krebse Ringer'sbuffer for 45 min, followed by a low-glucose buffer (3 mM glucose) for10 min, a high-glucose (17 mM glucose) buffer for 15 min, and finally alow-glucose buffer. Effluents were collected every 2 min starting fromthe first 3 mM medium. The medium samples or effluents collected in theexperiment was analyzed for insulin concentration using an Enzyme-LinkedImmunoSorbent Assay (ELISA) kit (Mercodia, Winston Salem, N.C.) forporcine insulin following the manufacturer's protocol.

Example 6 Representative Zwitterionic Copolymer Hydrogels: T CellEncapsulation

Functionalized star-shaped pCB polymers were synthesized via acombination of ATRP, “‘click” reaction, and conjugation. The synthesisis illustrated schematically in FIG. 7.

Difluorinated cyclooctyne moiety (DIFO3)-terminated pCB polymers withdifferent molecular weights were synthesized. pCB hydrogels were formedby mixing difluorinated cyclooctyne moiety-terminated pCB (pCB-A) withbiodegradable azide-GG-(KE)₂₀-GPQGIWGQ-(KE)₂₀-GG-azide together underphysiological conditions. The mechanical properties, equilibrium watercontent (EWC), and pore sizes of the hydrogel can be tuned by adjustingthe molecular weight of the polymers and the amount of used polymers.

Synthesis of Star pCB Polymer Via ATRP.

CBMA, 2,2′-bipyridine (bpy), catalysts, tetrafunctional initiator,pentaerythritoltetrakis(2-bromoisobutyrate) were placed in a 10 mLreaction tube, and the mixture was subjected to three freeze-pump-thawcycles. The mixture stayed under room temperature for 20 min, and waterand methanol were added at a 1:1 ratio. The reaction was allowed tocontinue at room temperature under stirring for 8 h. The polymer productwas recovered after treatment with alumina, and finally purified byprecipitation twice into acetone. The molecular weight of the polymercan be tuned by the stoichiometric ratio between the monomer and theinitiator. Star polymers with weight average molecular weights of 5000,20000 and 50000 were synthesized. Other living polymerization methodsuch as reversible addition-fragmentation chain-transfer (RAFT) can alsobe applied to synthesize the polymer.

Synthesis of Functionalized Star pCB Polymers Via Click Chemistry.

After purification, the terminal bromine groups of the star pCB wastransformed into azido groups by a nucleophilic substitution reactionwith sodium azide in water. The product was purified by dialysis.Lyophilization was used to remove the water. pCB-N₃ chains were thenreacted with the alkyne-NH₂ compounds in methanol with CuBr/PMDETA ascatalyst to produce the star pCB polymers with 4 arm numbers.Amine-terminated star-shaped pCB was obtained after purification viadialysis and lyophilization. Obtained polymer was further reacted withDIFO3 via EDC/NHS reaction and the resulted polymer was obtained afterpurification via dialysis and lyophilization.

Assessment of the Hydrogel Gelling Properties.

pCB gels were formed by adding DIFO3-terminated pCB with various polymerweight percentage solutions of aforementioned peptide in PBS at 37° C.The hydrogels obtained were washed thoroughly with distilled water toremove the unreacted polymers. The EWCs of the hydrogels were measuredat 37° C. using a gravimetric method. The temperature was controlled bya thermostatic water bath with a precision of ±0.1° C. The samples wereimmersed in 0.1 M PBS buffer solutions (pH 7.4) for at least 24 h andthen taken out, blotted with wet filter paper to remove water on thesurface, and weighed on a microbalance. After crosslinking, hydrogelswere allowed to freely swell in PBS for 24 hours.

In Vitro T Cell Encapsulation and Viability Test in pCB Hydrogel.

The biocompatibility of pCB hydrogels was tested in vitro. Humanperipheral blood CD4⁺CD45RA⁺ T Cells were added into the mixed solutionin the process of gelation. The T cell encapsulating hydrogel was placedand cultured in RPMI 1640 Medium+10% fetal bovine serum. After 7 daysculture, the hydrogel was dissolved by MMP protein and the cells wereharvested by centrifuge. The viability of the encapsulated T cellswithin pCB hydrogels were tested via trypan blue. Almost 100% viabilitywere found in the whole culture process (see FIG. 8).

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The invention claimed is:
 1. A hydrogel, comprising a first polymercovalently coupled to a second polymer, wherein the first polymercomprises a first core having two or more polymeric branches covalentlycoupled to and extending from the first core, wherein the polymericbranches covalently coupled to and extending from the first corecomprise first constitutional units selected from the group consistingof zwitterionic constitutional units and mixed charge constitutionalunits, and wherein the two or more polymeric branches covalently coupledto and extending from the first core each comprise one or more firstfunctional groups effective for covalently coupling the first polymer tothe second polymer, wherein the second polymer comprises a second corehaving two or more polymeric branches covalently coupled to andextending from the second core, wherein the polymeric branchescovalently coupled to and extending from the second core comprise secondconstitutional units selected from the group consisting of zwitterionicconstitutional units and mixed charge constitutional units, and whereinthe two or more polymeric branches covalently coupled to and extendingfrom the second core each comprise one or more second functional groupseffective for covalently coupling the second polymer to the firstpolymer; wherein the hydrogel comprises covalent bonds linking the firstpolymer and the second polymer formed by reaction of the one or morefirst functional groups and the one or more second functional groups;and wherein the one or more first functional groups and the one or moresecond functional groups are selected from an azide and an alkyne, anazide and an alkene, a thiol and a maleimide, or a thiol and adisulfide.
 2. A hydrogel, comprising a first polymer covalently coupledto a second polymer, wherein the first polymer comprises a first corehaving two or more polymeric branches covalently coupled to andextending from the first core, wherein the polymeric branches covalentlycoupled to and extending from the first core comprise firstconstitutional units selected from the group consisting of zwitterionicconstitutional units and mixed charge constitutional units, and whereinthe two or more polymeric branches covalently coupled to and extendingfrom the first core each comprise one or more first functional groupseffective for covalently coupling the first polymer to the secondpolymer, wherein the second polymer comprises a second core having twoor more polymeric branches covalently coupled to and extending from thesecond core, wherein the polymeric branches covalently coupled to andextending from the second core comprise second constitutional unitsselected from the group consisting of zwitterionic constitutional unitsand mixed charge constitutional units, and wherein the two or morepolymeric branches covalently coupled to and extending from the secondcore each comprise one or more second functional groups effective forcovalently coupling the second polymer to the first polymer; wherein thehydrogel comprises covalent bonds linking the first polymer and thesecond polymer via a crosslinking agent having two or more thirdfunctional groups, wherein the covalent bonds linking the first andsecond polymers are formed by reaction of the one or more firstfunctional groups and the two or more third functional groups and theone or more second functional groups and the two or more thirdfunctional groups, and wherein the one or more first functional groupsand the one or more second functional groups are independently selectedfrom an azide, alkyne, alkene, thiol, maleimide, or disulfide.
 3. Thehydrogel of claim 2, wherein the one or more first functional group is athiol, the one or more second functional group is a thiol, and the twoor more third functional groups are a thiol or a disulfide.
 4. Thehydrogel of claim 2, wherein the one or more first functional groups andtwo or more third functional groups are selected from an azide and analkyne, an azide and an alkene, a thiol and a maleimide, or a thiol anda disulfide.
 5. The hydrogel of claim 2, wherein the one or more secondfunctional groups and two or more third functional groups are selectedfrom an azide and an alkyne, an azide and an alkene, a thiol and amaleimide, or a thiol and a disulfide.
 6. The hydrogel of claim 1,wherein the first constitutional units are zwitterionic constitutionalunits.
 7. The hydrogel of claim 1, wherein the first constitutionalunits are mixed charge constitutional units.
 8. The hydrogel of claim 1,wherein the one or more first functional groups are positioned at theterminus of the polymeric branches covalently coupled to and extendingfrom the first core.
 9. The hydrogel of claim 1, wherein the one or morefirst functional groups are positioned along the backbone of thepolymeric branches covalently coupled to and extending from the firstcore.
 10. The hydrogel of claim 1, wherein the one or more of the firstconstitutional units comprise the one or more first functional groups.11. The hydrogel claim 1, wherein the second constitutional units arezwitterionic constitutional units.
 12. The hydrogel of claim 1, whereinthe second constitutional units are mixed charge constitutional units.13. The hydrogel of claim 1, wherein the one or more second functionalgroups are positioned at the terminus of the polymeric branchescovalently coupled to and extending from the second core.
 14. Thehydrogel of claim 1, wherein the one or more second functional groupsare positioned along the backbone of the polymeric branches covalentlycoupled to and extending from the second core.
 15. The hydrogel of claim1, wherein the one or more of the second constitutional units comprisethe one or more second functional groups.
 16. The hydrogel of claim 1,wherein the one or more first functional groups and the one or moresecond functional groups are the same.
 17. The hydrogel of claim 1,wherein the one or more first functional groups and the one or moresecond functional groups are different.
 18. The hydrogel of claim 1further comprising cells, viruses, bacteria, or components thereof, orgenetically altered variants thereof.
 19. The hydrogel of claim 18,wherein the cells are exocrine secretory epithelial cells, hormonesecreting cells, keratinizing epithelial cells, wet stratified barrierepithelial cells, sensory transducer cells, autonomic neuron cells,sense organ cells, peripheral neuron supporting cells, neurons, glialcells, lens cells, hepatocytes, adipocytes, lipocytes, barrier functioncells, kidney cells, heart cells, extracellular matrix cells,contractile cells, blood cells, immune system cells, erythrocytes,monocytes, neutrophils, mast cells, T cells, stem cells, germ cells,nurse cells, interstitial cells, progenitor cells, or hematopoietic stemcells.
 20. The hydrogel of claim 1, wherein the one or more firstfunctional group is a thiol and the one or more second functional groupis a thiol, maleimide, or disulfide.
 21. The hydrogel of claim 1,wherein the one or more first functional group is an azide and the oneor more second functional group an alkyne or an alkene.
 22. The hydrogelclaim 2, wherein the first constitutional units are zwitterionicconstitutional units.
 23. The hydrogel of claim 2, wherein the firstconstitutional units are mixed charge constitutional units.
 24. Thehydrogel of claim 2, wherein the one or more first functional groups arepositioned at the terminus of the polymeric branches covalently coupledto and extending from the first core.
 25. The hydrogel of claim 2,wherein the one or more first functional groups are positioned along thebackbone of the polymeric branches covalently coupled to and extendingfrom the first core.
 26. The hydrogel of claim 2, wherein one or more ofthe first constitutional units comprise the one or more first functionalgroups.
 27. The hydrogel of claim 2, wherein the second constitutionalunits are zwitterionic constitutional units.
 28. The hydrogel of claim2, wherein the second constitutional units are mixed chargeconstitutional units.
 29. The hydrogel of claim 2, wherein the one ormore second functional groups are positioned at the terminus of thepolymeric branches covalently coupled to and extending from the firstcore.
 30. The hydrogel of claim 2, wherein the one or more secondfunctional groups are positioned along the backbone of the polymericbranches covalently coupled to and extending from the first core. 31.The hydrogel of claim 2, wherein the one or more of the secondconstitutional units comprise the one or more second functional groups.32. The hydrogel of claim 2, wherein the one or more first and the oneor more second functional groups are the same.
 33. The hydrogel of claim2, wherein the one or more first and the one or more second functionalgroups are different.
 34. The hydrogel of claim 2, further comprisingcells, viruses, bacteria, or components thereof, or genetically alteredvariants thereof.
 35. The hydrogel of claim 34, wherein the cells areexocrine secretory epithelial cells, hormone secreting cells,keratinizing epithelial cells, wet stratified barrier epithelial cells,sensory transducer cells, autonomic neuron cells, sense organ cells,peripheral neuron supporting cells, neurons, glial cells, lens cells,hepatocytes, adipocytes, lipocytes, barrier function cells, kidneycells, heart cells, extracellular matrix cells, contractile cells, bloodcells, immune system cells erythrocytes, monocytes, neutrophils, mastcells, T cells, stem cells, germ cells, nurse cells, interstitial cells,progenitor cells, or hematopoietic stem cells.